Anti-trypanosomal peptides and uses thereof

ABSTRACT

The present invention provides methods of killing, inhibiting the growth, and/or inhibiting the reproduction of kinetoplastid protozoan with hydrophobic signal sequence peptides and compositions including such hydrophobic signal sequence peptides.

CONTINUING APPLICATION DATA

This application is a continuation-in-part of International ApplicationNo. PCT/US2010/032545, filed Apr. 27, 2010, which claims the benefit ofU.S. Provisional Application Ser. Nos. 61/172,908, filed Apr. 27, 2009,and 61/317,895, filed Mar. 26, 2010, all of which are incorporatedherein by reference in their entireties.

GOVERNMENT FUNDING

The present invention was made with government support under Grant Nos.AI039033 and 1F32AI080114-01A1, awarded by the National Institutes ofHealth. The Government has certain rights in this invention.

BACKGROUND

Trypanosoma brucei is the causative agent of both a veterinary wastingdisease and human African trypanosomiasis, or sleeping sickness. HumanAfrican trypanosomiasis occurs in 36 countries in sub-Saharan Africa,threatening an estimated 60 million people with debilitating disease.Currently, approximately 500,000 people are infected with Africantrypanosomes. No vaccines are available for prevention of infection byT. brucei. Without chemotherapeutic treatment, T. brucei kills infectedhumans. Treatment of infected individuals is limited and there is anincrease in the number of relapses from established drug treatment.Drugs currently in use are quite toxic and cause serious side effectsand in some cases death. And, the development of drug resistance is ofconcern. Few drug candidates are currently under clinical trials, andone of the more promising compounds, DB75, has already shown a markedtendency to induce drug resistance (Lanteri et al., 2006, Mol Pharmacol;70(5):1585-1592). Consequently, there is a strong need for new and saferdrugs for the treatment of trypanosomiasis and new drugs must bedeveloped in order to prepare for possible emergence of drug resistancein the parasites.

SUMMARY OF THE INVENTION

The present invention includes methods of killing a bloodstream form(BSF) of a kinetoplastid protozoan of the genus Trypanosoma, the methodincluding contacting the protozoan with a hydrophobic signal sequencepeptide, wherein the hydrophobic signal sequence peptide consists ofabout 12 to about 25 amino acid residues, and wherein the hydrophobicsignal sequence peptide contains a positively charged amino acid atposition minus five relative to the C-terminus of the hydrophobic signalsequence peptide.

The present invention includes methods of treating or preventing atrypanosomal infection in a subject, the method including administeringto the subject an effective amount of a hydrophobic signal sequencepeptide, wherein the hydrophobic signal sequence peptide consists ofabout 12 to about 25 amino acid residues, and wherein the hydrophobicsignal sequence peptide contains a positively charged amino acid atposition minus five relative to the C-terminus of the hydrophobic signalsequence peptide.

In some aspects of the methods of the present invention, the hydrophobicsignal sequence peptide has the sequence of an uncleaved signal sequencepeptide of haptoglobin-related protein or paraoxonase 1.

In some aspects of the methods of the present invention, the hydrophobicsignal sequence peptide is soluble in ethanol or dimethyl sulfoxide(DMSO).

In some aspects of the methods of the present invention, the hydrophobicsignal sequence peptide includes at least nine consecutive amino acidresidues of MSDLGAVISLLLWGRQLFA (SEQ ID NO:1), MAKLIALTLLGMGLALFRNHQS(SEQ ID NO:3), a derivative of SEQ ID NO:1, or a derivative of SEQ IDNO:3, wherein a derivative of SEQ ID NO:1 or SEQ ID NO:3 has up to fourhydrophobic amino acid residues of SEQ ID NO:1 or SEQ ID NO:3 exchangedfor another hydrophobic amino acid, and/or up to four positively chargedamino acid residues of SEQ ID NO:1 or SEQ ID NO:3 exchanged for anotherpositively charged amino acid.

In some aspects of the methods of the present invention, the hydrophobicsignal sequence peptide is selected from the group consisting ofMSDLGAVISLLLWGRQLFA (SEQ ID NO:1), SDLGAVISLLLWGRQLFA (SEQ ID NO:2),MAKLIATLLGMGLALFRNHQS (SEQ ID NO:3), AKLIATLLGMGLALFRNHQS (SEQ ID NO:4),or SDLGAVISLLWGRQLFA (SEQ ID NO:7), WDLGAVISLLLGGRQLFA (SEQ ID NO:15),SDLGAVIWLLLGGRQLFA (SEQ ID NO:16) and SDLGAVISLLLGGRQLFW (SEQ ID NO:17).

In some aspects of the methods of the present invention, the trypanosomeis selected from the group consisting of Trypanosoma brucei brucei, T.b. gambiense, and T. b. rhodesiense, T. congolense, and T. vivax.

In some aspects of the methods of the present invention, the hydrophobicsignal sequence peptide is administered as a composition furtherincluding liposome, emulsion, or micelle.

In some aspects of the methods of the present invention, the hydrophobicsignal sequence peptide is administered as a composition furtherincluding an RNA aptamer that binds to a structurally conserved regionof a trypanosome variant surface glycoprotein (VSG).

The present invention includes compositions including a hydrophobicsignal sequence peptide and a pharmaceutical carrier suitable forparenteral or enteral administration to a mammal, wherein thehydrophobic signal sequence peptide consists of about 12 to about 25amino acid residues, and wherein the hydrophobic signal sequence peptidecontains a positively charged amino acid at position minus five relativeto the C-terminus of the hydrophobic signal sequence peptide.

The present invention includes methods of killing a bloodstream form ofa kinetoplastid protozoan of the genus Trypanosoma, the method includingcontacting the protozoan with a composition of claim 11.

The present invention includes methods of treating or preventing atrypanosomal infection in a subject, the method including administeringto the subject an effective amount of a composition of claim 11.

In some aspects of the compositions of the present invention, thecomposition is pyrogen-free.

In some aspects of the compositions of the present invention, thehydrophobic signal sequence peptide has the sequence of an uncleavedsignal sequence peptide of haptoglobin-related protein or paraoxonase 1.

In some aspects of the compositions of the present invention, thehydrophobic signal sequence peptide is soluble in ethanol or dimethylsulfoxide (DMSO).

In some aspects of the compositions of the present invention, thehydrophobic signal sequence peptide includes at least nine consecutiveamino acid residues of MSDLGAVISLLLWGRQLFA (SEQ ID NO:1),MAKLIALTLLGMGLALFRNIHQS (SEQ ID NO:3), a derivative of SEQ ID NO:1, or aderivative of SEQ ID NO:3, wherein a derivative of SEQ ID NO:1 or SEQ IDNO:3 has up to four hydrophobic amino acid residues of SEQ ID NO:1 orSEQ ID NO:3 exchanged for another hydrophobic amino acid, and/or up tofour positively charged amino acid residues of SEQ ID NO:1 or SEQ IDNO:3 exchanged for another positively charged amino acid.

In some aspects of the compositions of the present invention, thehydrophobic signal sequence peptide is selected from the groupconsisting of MSDLGAVISLLLWGRQLFA (SEQ ID NO:1), SDLGAVISLLLWGRQLFA (SEQID NO:2), MAKLIATLLGMGLALFRNHQS (SEQ ID NO:3), AKLIATLLGMGLALFRNHQS (SEQID NO:4), or SDLGAVISLLWGRQLFA (SEQ ID NO:7), WDLGAVISLLLGGRQLFA (SEQ IDNO:15), SDLGAVIWLLLGGRQLFA (SEQ ID NO:16) and SDLGAVISLLLGGRQLFW (SEQ IDNO:17).

In some aspects of the compositions of the present invention, thecomposition includes a liposome, emulsion, or micelle including thehydrophobic signal sequence peptide.

In some aspects of the compositions of the present invention, furtherincluding an RNA aptamer that binds to a structurally conserved regionof a trypanosome variant surface glycoprotein (VSG).

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the killing of T. b. brucei by the Hpr-SP. In FIG. 1A, wildtype 427 T. b. brucei cells were incubated with increasingconcentrations of Hpr-SP (8-80 μM) added from a stock solution in DMSO.Positive controls were trypanosome lytic factor (TLF). Addition ofequivolume DMSO or equimolar concentrations of a non-specifichydrophilic 19 amino acid peptide (NS 19mer; SEQ ID NO:10) gave notrypanolysis. The inset in FIG. 1A shows cells treated with Hpr-SP. FIG.1B is a cartoon of Hpr and the derived peptide Hpr-SP (SEQ ID NO:1).Hydrophobic residues are in bold.

FIG. 2 shows the results of lysis assays conducted with T. b. bruceiexpressing SRA or procyclic form T. b. brucei.

FIG. 3 presents the results of lysis assays in the presence ofchloroquine, NH₄Cl or at 3° C.

FIG. 4 shows modeling of T. b. brucei cellular membranes with liposomes.Artificial vesicles were constructed with the phosphatidylcholinescorresponding in acyl chain composition to either the VSG or procyclinlipid anchors, egg phosphatidylcholine (egg PC, transitiontemperature<0° C.) for VSG and distearoylphosphatidylcholine (DSPS) forprocyclin. The leakage of liposomally entrapped calcein wasfluorescently monitored as an indicator of Hpr-SP membrane interaction.

FIG. 5 monitors membrane rigidity in live T. b. brucei with theanisotropic probe TMA-DPH. The addition of Hpr-SP results in an increasein the anisotropy of TMA-DPH labeled cells, indicating membranerigidification. Equivolmes of DMSO, the Hpr-SP solvent, results ineither no change (0.167% DMSO corresponding to 8 μM Hpr-SP) or adecrease (0.4% DMSO corresponding to 20 μM Hpr-SP) in membrane rigidity.

FIG. 6 assayed cell viability via trypan blue exclusion. Human embryonickidney cells were incubated with increasing concentrations of Hpr-Sp fortwo hours at 37° C. No cell death was observed with trypanolyticconcentrations of Hpr-SP or equivolume addition of DMSO. Positivekilling controls provided by the lytic peptide melittin.

FIG. 7 demonstrates toxicity and specificity of SHP-1 for BSF Africantrypanosomes. In FIG. 7A, SHP-1 was assayed for killing activity againstbloodstream form (BSF) T. b. brucei (diamond), T. b. rhodesiense(square), T. b. gambiense (triangle) and procyclic form (PCF) T. b.brucei (circle). Equivolume additions of solvent DMSO (black circle)showed no toxic effect. FIG. 7B is DIC micrographs of untreated, T. b.brucei fixed with formaldehyde and unfixed T. b. brucei incubated with 4and 80 μM SHP-1 for two hours at 37° C. FIG. 7C presents binding ofTexas-red labeled SHP-1 to bloodstream form (BSF), with and withoutpeptide, and procyclic form (PCF), with and without peptide. T. b.brucei monitored by flow cytometry.

FIG. 8 demonstrates that SHP-1 is not toxic to mammalian cells. In FIG.8A, human cell lines, HEK (circles) and LNCaP (square), are not killedby SHP-1 as assayed by trypan blue exclusion. FIG. 8B demonstrates thatSHP-1 (80 μM) does not induce morphological changes in HEK or LNCaPcells. The cytolytic peptide melittin (25 μM) was utilized as a positivecontrol. FIG. 8C, flow cytometry indicates that Texas-red labeled SHP-1does not bind HEK or LNCaP cells. FIG. 8D, potential hemolytic activityof SHP-1 was assayed against fresh human erythrocytes.

FIG. 9 presents hydrophobicity requirements for trypanosome killing bysmall peptides. FIG. 9A presents peptide amino acid sequences, N toC-terminal. SBP-1 is SDLGAVISLLLWGRQLFA (SEQ ID NO:2); SHP-2 isAKLIALTLLGMGLALFRNHQS (SEQ ID NO:4); SHP-1-ΔL is SDLGAVISLLWGRQLFA (SEQID NO:7); SHP-1-ΔLLL is SDLGAVISWGRQLFA (SEQ ID NO:8); SBP-1-ΔLGA isSDVISLLLWGRQLFA (SEQ ID NO:9); and NSP is ERTEESWGRRFWRRGEAC (SEQ IDNO:10). FIG. 9B demonstrates the killing of bloodstream form (BSF) T. b.brucei by SHP-1 (squares) and its variants, SHP-2 (diamonds), SHP-1-ΔL(white circles), SHP-1ΔLLL (black triangles), SHP-1-ΔLGA (graytriangles) and a non-specific hydrophilic peptide (NSP, gray circles).FIG. 9C is representative traces of liposome permeabilization by 200 nMSHP-1, 200 nM SHP-2 and the deletion variants, 500 nM SHP-1-ΔL, 500 nMSHP-1-ΔLLL and 500 nM SHP-1Δ-LGA.

FIG. 10 demonstrates that SHP-1 acts at the surface of bloodstream form(BSF) African trypanosomes. FIG. 10A demonstrates that killing of BSF T.b. brucei does not require cellular uptake, indicated by robustSHP-1-mediated killing at 3° C., a temperature non-permissive forendocytosis. Alexa 594-labeled transferrin (Tf) binds at the flagellarpocket, but is not taken up at 3° C. (inset). In FIG. 10B, fluorescencemicroscopy with Texas-red labeled SHP-1 reveals diffuse labeling of thecell surface rather than accumulation within an intracellular vesicle asdisplayed by Alexa 594-labeled transferrin (Tf). Cell nuclei andkinetoplast are labeled with DAPI.

FIG. 11 shows that SHP-1 interacts with membranes exhibiting fluid phasepacking. FIG. 11A demonstrates that lateral van der Waals interactionsdictate sensitivity to Hpr-SP, as indicated by the ability of 200 nMSHP-1 to readily elicit calcein leakage from unilamellar liposomescomposed of egg phosphatidylcholine (PC), a highly fluid, heterogeneousmixture of naturally derived lipids with T_(m)<0° C., whereas liposomescomposed of homogenous lipid species with symmetric acyl chains, 15:0(T_(m)=34° C.) or 16:0 (T_(m)=41° C.) are resistant. No permeabilizationis seen against liposomes composed of symmetric 17:0 or 18:0 PC either.In FIG. 11B, the addition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) (indicated by percentage) to refractory compositions1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) renders liposomessusceptible to permeabilization by SHP-1. FIG. 11C demonstrates thatliposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine(SOPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),asymmetrical PC lacking myristate, but exhibiting fluid lipid packingdue to unsaturations (16:0, 18:1, T_(m)=−2° C. and 18:0, 18:1, T_(m)=6°C.) are susceptible to permeabilization by SHP-1. FIG. 11D demonstratesthat thermal fluidization of symmetrical 15:0 and 16:0 PC lipid bilayersrenders liposomes susceptible to permeabilization by SHP-1. Assays werecarried out at approximately 60° C.

FIG. 12 demonstrates that SHP-1 changes the mechanoelastic properties ofbloodstream form (BSF) T. brucei membranes and induces dramatic changesin motility. In FIG. 12A, the rigidity of BSF T. brucei plasma membranesis increased by the addition of SHP-1 as revealed by the anisotropicchanges in the membrane probe1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatrienep-toluenesulfonate (TMA-DPH) (SHP-1—closed circle, DMSO only—opencircles). Consistent with the lack of binding to more rigid lipidbilayers and the higher anisotropic values acquired for procyclic form(PCF) T. brucei, SHP-1 has no effect on the anisotropy of PCF T. bruceicell plasma membranes (SHP-1—closed squares, DMSO only—open squares).FIG. 12B depicts the changes in motility of bloodstream form T. bruceitreated with 8 M FIG. 12C presents images captured via DICvideomicroscopy illustrate the normal, hyperactive and constrictedphenotype of cells treated with Hpr-SP. The normal motion of bloodstreamform T. brucei displays a full rotation within 100 ms. Hyperactive cellsappear to have accomplished 1.5-2 rotations within 50 ms. Theconstricted phenotype is commonly seen as a bent or boomerang shapedtrypanosome that fails to complete a rotation.

FIG. 13 demonstrates small hydrophobic peptide mediated killing ofAfrican trypanosomes. In FIG. 13A the metacyclic developmental form (thedevelopmental stage injected during a tsetse fly bite) of T. b. brucei(circles) and the veterinary pathogenic African trypanosomes, T. vivax(triangles) and T. congolense (squares), were assayed for susceptibilityto SHP-1 in a two hour in vitro killing assay. In FIG. 13B the sequencesof trypanolytic and non-trypanolytic SHP are shown from N- to C-terminusand aligned to the C-terminus in order to emphasize the identity of theamino acid at position −5 relative to the putative signal peptidasecleavage site. Positively charged amino acids are circled, negativelycharged amino acids are boxed and non-polar amino acids are underlined.SHP-1 is SDLGAVISLLLWGRQLFA (SEQ ID NO:2); SHP-2 isAKLIALTLLGMGLALFRNHQS (SEQ ID NO:4); SHP-3 is FHQIWAALLYFYGIILNSIY (SEQID NO:11); SHP-3ΔR is FHQIWAALLYFYGERNSIY (SEQ ID NO:12); SHP-3ΔE isFHQIWAALLYFYGMENSIY (SEQ ID NO:13); and SHP-1swap is SRLGAVISLLLWGDQLFA(SEQ ID NO:14). In FIG. 13C the SHP listed in FIG. 13B were testedagainst BSF T. b. brucei in a two hour killing assay. Peptide used areshown in FIG. 13B; SHP-1 (closed circles), SHP-3 (closed squares),SHP-3ΔR (closed triangles), SHP-3ΔE (open triangles), SHP-1swap (opencircles).

FIG. 14 presents membrane rigidity changes and physiologicalconsequences of SHP. The rigidity of the interior (FIG. 14A) orinterfacial (FIG. 14B) region of the plasma membrane of BSF T. b. bruceitreated with increasing concentrations of SHP-1 (closed circles), SHP-3(closed squares), SHP-3ΔR (closed triangles), SHP-3ΔE (open triangles),SHP-1swap (open circles) or solvent alone (DMSO) (X) was determined bymeasuring the fluorescence depolarization of DPH or TMA-DPHrespectively. FIG. 14C is a FRAP analysis of the mobile fraction of BSFT. b. brucei VSG in the presence (gray bars) or absence (black bars) of8 μM SHP-1. In FIG. 14D live BSF T. b. brucei treated with 40 μM SHP-1,SHP-3 or SHP-3ΔR were scored via video microscopy for hyperactivation(white bars), constriction (gray bars) or normal motility (black bars).

FIG. 15 demonstrates orientation of trypanocidal and non-trypanocidalSHP in lipid bilayers. The depth of tryptophan penetration into thehydrocarbon region of model liposomes was determined via parallaxanalysis. Assuming a hydrocarbon bilayer thickness of 29 Å, andutilizing depth values from tryptophans substituted along the entirepeptides, SHP-1 (circles) and SHP-3 (squares) are illustrated in thecontext of a helical peptide embedded in the outer leaflet of aphospholipid bilayer.

FIG. 16 shows SHP binding to BSF T. b. brucei. FITC-labeled SHP-1 andSHP-3 were assayed for binding to BSF T. b. brucei via flow cytometry.Trypanosomes were adjusted to 3×10⁶ cells/ml in HMI 9 media with 10%fetal bovine serum, 8 μM FITC-SHP-1 or FITC-SHP-3 was added and 50,000cells were immediately counted.

FIG. 17 shows trypanosome killing and membrane interaction with SHPtryptophan variants. In FIG. 17A small hydrophobic peptide-1 tryptophanvariants SHP-1Δ W1 (●), SHP-1ΔW8 (□) and SHP-1ΔW18 (▴) were tested fortrypanocidal activity. In FIG. 17B the ability of the SHP-1 tryptophanvariants 1 μM SHP-1ΔW1, 1 SHP-1ΔW8, 0.2 μM SHP-1 and 0.2 μM SHP-1ΔW18 tointeract with lipid bilayers was determined by monitoring the release ofentrapped calcein from unilamellar egg phosphatidylcholine liposomes. InFIG. 17C the ability of the SHP-3 tryptophan variants 1 μM SHP-3ΔW1, 1μM SHP-3, 1 μM SHP-3ΔW13 and 4 μM SHP-3ΔW20 to interact with lipidbilayers was determined by monitoring the release of entrapped calceinfrom unilamellar egg phosphatidylcholine liposomes.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The present invention demonstrates, for the first time, the trypanocidalactivity of hydrophobic signal sequence peptides and provides methods ofkilling, inhibiting the growth, and/or inhibiting the reproduction ofkinetoplastid protozoan, including, but not limited to, kinetoplastidprotozoan of the genus Trypanosoma, with hydrophobic signal sequencepeptides and compositions including such hydrophobic signal sequencepeptides. Protozoal pathogens have a worldwide impact and causesymptomatic as well as asymptomatic infections. Unfortunately, effectivetreatments for the different diseases are by and large not available.This is especially true for African trypanosomiasis, a parasitic diseasecaused by a specific class of protozoan organisms, trypanosomes. Theeukaryotic parasite Trypanosoma brucei infects mammals and causesdebilitating diseases in both humans and cattle. The subspeciesTrypanosoma brucei brucei (T. b. brucei) causes the wasting disease incattle nagana in cattle, while the two subspecies Trypanosoma bruceirhodesiense (T. b. rhodesiense) and Trypanosoma brucei gambiense (T. b.gambiense) cause sleeping sickness, also known as human Africantrypanosomiasis (HAT), in humans.

The methods and compositions of the present invention are applicable fora variety of protozoa, including, but not limited to, kinetoplastidprotozoa. Kinetoplastids are a group of flagellate protozoa, including anumber of parasites responsible for serious diseases in humans and otheranimals, including economically relevant livestock, as well as variousforms found in soil and aquatic environments. They are included in theEuglenozoa, and are distinguished from other such forms mainly by thepresence of a kinetoplast, a DNA-containing granule located within thesingle mitochondrion and associated with the flagellar bases.Kinetoplastids typically have complex life-cycles involving more thanone host, and go through various morphological stages. The mostdistinctive of these is the trypomastigote stage, where the flagellumruns along the length of the cell and is connected to it by anundulating membrane. Kinetoplastid protozoa, include, for example,protozoa of the Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas,Leishmania, Leptomonas, Phytomonas, Trypanosoma, and Wallaceina genera.Diseases caused by trypanosomes include African Sleeping Sickness andSouth American Chagas Disease, from species of Trypanosoma, andleishmaniasis, from species of Leishmania.

In some embodiments, the methods and compositions of the presentinvention are applicable for species of Trypanosoma, including, but arenot limited to, T. avium, which causes trypanosomiasis in birds, T.boissoni, T. brucei, which causes sleeping sickness in humans and naganain cattle, T. carassii, in freshwater teleosts, T. cruzi, which causesChagas disease in humans, T. gambiense, T. rhodesiense, T. congolense,which causes nagana in cattle, horses, and camels, T. equinum, T.equiperdum, which causes dourine or covering sickness in horses, T.evansi, which causes one form of the disease surra in certain animals,T. lewisi, in rats, T. melophagium, T. percae in fish, T. rangeli, T.rotatorium in amphibian, T. simiae, T. suis, T. theileri, T. triglae,and T. vivax.

In some embodiments, the methods and compositions of the presentinvention are applicable for the protozoan T. brucei, including, but notlimited to, Trypanosoma brucei brucei, T. cruzi, T. brucei, T. b.gambiense, T. b. rhodesiense, T. congolense, and T. vivax. In someembodiments, the methods and compositions of the present invention areapplicable for the protozoan Trypanosoma brucei brucei, T. b. gambiense,and T. b. rhodesiense, T. congolense, and T. vivax. In some embodiments,the methods and compositions of the present invention are applicable forvarious protozoan Trypanosoma brucei, excluding T. cruzi.

The present invention has identified peptides that demonstrate theability of killing the bloodstream form (BSF) of kinetoplastidprotozoans of the genus Trypanosoma. More specifically, thesetrypanocidal peptides are based upon the hydrophobic N-terminal signalsequences of various mammalian plasma proteins. Additionally thesepeptides exhibit reduced or no toxicity towards mammalian cell lines andinduce limited or no hemolysis.

Proteins have intrinsic signals that govern their transport andlocalization within the cell. Almost all proteins that are transportedto the endoplasmic reticulum and destined either to be secreted or to bemembrane components have a short signal sequence that directs thetransport of a protein through the cell membrane. This signal peptide isan amino acid sequence present on the protein. It is usually at the Nterminus. It is usually is cleaved off and absent in the mature protein,though in some proteins, the signal peptide uncleaved and is retained inthe mature protein. Signal peptides are highly hydrophobic but with somepositively charged residues.

A trypanocidal peptides of the present invention may also be referred toherein as an anti-trypanosomal peptides, a signal peptide, SP, a signalsequence peptide, a hydrophobic peptide, a hydrophobic signal sequencepeptide, a N-terminal signal sequence, or a hydrophobic N-terminalsignal sequence.

A trypanocidal peptide of the present invention may be a knownhydrophobic N-terminal signal sequence, or a fragment or derivativethereof. A trypanocidal peptide of the present invention may be ahydrophobic N-terminal signal sequence, with or without the N-terminalmethionine residue, or a derivative or fragment thereof. Derivativesinclude, but are not limited to, the exchange of one, two, three, four,or more hydrophobic amino acid residues for another hydrophobic aminoacid. Such hydrophobic amino acids include valine (“Val” or “V”),isoleucine (“He” or “I”), leucine (“Leu” or “L”), methionine (“Met” or“M”), phenyalanine (“Phe” or “F”), tryptophan (“Trp” or “W”), cysteine(“Cys” or “C”), alanine (“Ala” or “A”), tyrosine (“Tyr” or “Y”),histidine (“His” or “H”), threonine, (“Thr” or “T”), serine (“Ser” or“S”), proline (“Pro” or “P”), glycine (“Gly” or “G”), arginine (“Arg” or“R”), and lysine (“Lys” or “K”). In some embodiments, derivativesinclude, but are not limited to, the exchange of one, two, three, four,or more very hydrophobic amino acid residues for another veryhydrophobic amino acid. Such very hydrophobic amino acids include valine(“Val” or “V”), isoleucine (“Ile” or “I”), leucine (“Leu” or “L”),methionine (“Met” or “M”), phenyalanine (“Phe” or “F”), tryptophan(“Trp” or “W”), and cysteine (“Cys” or “C”). Derivatives include, butare not limited to, the exchange of one, two, three, four, or morepositively charged amino acid residues for another positively chargedamino acid. Derivatives include, but are not limited to, the deletion ofone, two, three, or more hydrophobic amino acids from the known signalsequence. Derivatives also include, but are not limited to, the deletionof one, two, three, or more very hydrophobic amino acids from the knownsignal sequence. Derivatives may also include any combination of theabove substitutions and/or deletions. Fragments may include, but are nolimited to, peptides having at least 5, at least 6, at least 7, at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, or at least 22 consecutive amino acids ofthe hydrophobic N-terminal signal peptide. Such sequences may beconsecutive sequences.

A trypanocidal peptide of the present invention may be any of a varietyof lengths. A trypanocidal peptide of the present invention may be, forexample, about 3 to about 60 amino acids in length, about 9 to about 25amino acids in length, about 10 to about 25 amino acids in length, about12 to about 25 amino acids in length, about 14 to about 25 amino acidsin length, about 17 to about 25 amino acids in length, about 19 to about25 amino acids in length, about 19 to about 22 amino acids in length, orabout 17 to about 23 amino acids in length. A trypanocidal peptide ofthe present invention may be, for example, at least about 9, at leastabout 10, at least about 11, at least about 12, at least about 13, atleast about 14, at least about 15, at least about 16, at least about 17,at least about 18, at least about 19, at least about 20, at least about21, at least about 22, at least about 23, at least about 24, or at leastabout 25 amino acids in length and/or no more that about 10, no morethan about 11, no more than about 12, no more than about 13, no morethan about 14, no more than about 15, no more than about 16, no morethan about 17, no more than about 18, no more than about 19, no morethan about 20, no more than about 21, no more than about 22, no morethan about 23, no more than about 24, no more than about 25, no morethan about 26, no more than about 27, no more than about 28, no morethan about 29, or no more than about 30 amino acids in length. Forexample, a trypanocidal peptide of the present invention may be, forexample, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, or about 30amino acids in length. For example, a trypanocidal peptide of thepresent invention may be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 amino acids in length.

A trypanocidal peptide of the present invention may be a hydrophobicpeptide. For example, in a hydrophobic peptide at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, or at least about 95% of the amino acid residues arehydrophobic amino acid residues. For example, in a hydrophobic peptideall but 10 or fewer, all but 9 or fewer, all but 8 or fewer, all but 7or fewer, all but 6 or fewer, all but 5 of fewer, all but 4 or fewer,all but 3 or fewer, all but 2 or fewer, or all but 1 of the amino acidresidues are hydrophobic amino acid residues.

A trypanocidal peptide of the present invention may have a positivelycharged amino acid residue at a position minus 5 from the C-terminus ofthe peptide. Such positively charged amino acids include arginine (“Arg”or “R”), lysine (“Lys” or “K”), and histidine (“His” or “H”).

A trypanocidal peptide of the present invention may have an alphahelical conformation in a nonpolar environment or solution.

Trypanocidal peptides of the present invention may be identified by anyof a variety of means, including, but not limited to, any of the assaysdescribed herein, including, but not limited to, a trypanosome lysisassay. Trypanocidal peptides of the present invention may target thecell membrane of protozoans, including, but not limited to kinetoplastidprotozoans of the genus Trypanosoma, and may that increase the rigidityof lipid bilayers of the cell membrane. Trypanocidal peptides of thepresent invention may selectively partition into the plasma membrane ofBSF trypanosomes resulting in an increase in the rigidity of thebilayer, changes in cell motility and/or subsequent cell death.

A trypanocidal peptide of the present invention may be a hydrophobicN-terminal signal sequence of a protein that is associated with highdensity lipoproteins (HDL). A trypanocidal peptide of the presentinvention may be a hydrophobic N-terminal signal sequence of anapolipoprotein, including, but not limited to a human apolipoprotein.

In some embodiments, a trypanocidal peptide of the present invention isan uncleaved, hydrophobic N-terminal signal peptide, that is, a signalpeptide that is retained on the mature protein, and derivatives andfragments thereof. Examples of proteins with uncleaved signal peptidesare haptoglobin-related protein (Hpr), serum paraoxonase/arylesterase(PON1), and apolipoprotein M (Apo M), including, but not limited to,human haptoglobin-related protein, human serum paraoxonase/arylesterase(PON1), and human apolipoprotein M (Apo M).

In some embodiments, a trypanocidal peptide of the present inventionincludes the hydrophobic N-terminal signal peptide ofhaptoglobin-related protein (also referred to herein as “Hpr” or “HPR”),including, but not limited to, human haptoglobin-related protein(UniProtKB/Swiss-Prot Accession No: P00739), fragments and derivativesthereof. Hpr is an integral part of two distinct high molecular weightcomplexes (trypanosome lytic factor 1 (TLF1) and trypanosome lyticfactor 2 (TLF2)) that are lytic for the African parasite Trypanosomabrucei brucei. See, for example, Smith et al., 1995, Science;268(5208):284-6; Drain et al., 2001, J Biol Chem; 276(32):30254-60; andMuranjan et al., 1998, J Biol Chem; 273(7):3884-7. Humanhaptoglobin-related protein demonstrates an uncleaved signal peptide.For example, trypanocidal peptides of the present invention includepeptides having the N-terminal amino acid residues 1-19, or 2-19 of thehuman haptoglobulin-related protein (MSDLGAVISLLLWGRQLFA (SEQ ID NO:1)and SDLGAVISLLLWGRQLFA (SEQ ID NO:2), respectively), fragments, andderivatives thereof. Derivatives include any of the derivativesdescribed herein, including, but are not limited to, the exchange ofone, two, three, four, or more hydrophobic amino acid residues foranother hydrophobic amino acid. Derivatives may include, but are notlimited to, the exchange of one, two, three, four, or more veryhydrophobic amino acid residues for another very hydrophobic amino acid.Derivatives include, but are not limited to, the exchange of one, two,three, four, or more positively charged amino acid residues for anotherpositively charged amino acid, including, but not limited to, asubstitution at the positively charged amino acid at position minus 5from the C terminus. Derivatives may include, but are not limited to,the deletion of one, two, three, or more hydrophobic amino acids fromthe known signal sequence. Derivatives may also include, but are notlimited to, the deletion of one, two, three, or more very hydrophobicamino acids from the known signal sequence. Derivatives also include anycombination of the above substitutions and/or deletions. Fragments mayinclude, but are no limited to, peptides having at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, or at least 22consecutive amino acids of the hydrophobic N-terminal signal peptide ofhaptoglobin-related protein or derivative thereof, including, but notlimited to, human haptoglobin-related protein (UniProtKB/Swiss-ProtAccession No: P00739)

In some embodiments, a trypanocidal peptide of the present inventionincludes the hydrophobic N-terminal signal peptide of serumparaoxonase/arylesterase (PON1), including, but not limited to, humanserum paraoxonase/arylesterase (UniProtKB/Swiss-Prot Accession No.P27169), fragments and derivatives thereof. Serumparaoxonase/arylesterase, like haptoglobin-related protein, demonstratesan uncleaved signal peptide. For example, trypanocidal peptides of thepresent invention include peptides having the N-terminal amino acidresidues 1-22, or 2-22 of human paraoxonase-1 protein(MAKLIALTLLGMGLALFRNHQS (SEQ ID NO:3) and AKLIALTLLGMGLALFRNHQS (SEQ IDNO:4), respectively), fragments, and derivatives thereof. Derivativesinclude any of the derivatives described herein, including, but are notlimited to, the exchange of one, two, three, four, or more hydrophobicamino acid residues for another hydrophobic amino acid. Derivatives mayinclude, but are not limited to, the exchange of one, two, three, four,or more very hydrophobic amino acid residues for another veryhydrophobic amino acid. Derivatives include, but are not limited to, theexchange of one, two, three, four, or more positively charged amino acidresidues for another positively charged amino acid, including, but notlimited to, a substitution at the positively charged amino acid atposition minus 5 from the C terminus. Derivatives may include, but arenot limited to, the deletion of one, two, three, or more hydrophobicamino acids from the known signal sequence. Derivatives may alsoinclude, but are not limited to, the deletion of one, two, three, ormore very hydrophobic amino acids from the known signal sequence.Derivatives also include any combination of the above substitutionsand/or deletions. Fragments may include, but are no limited to, peptideshaving at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, or at least 22 consecutive amino acids of the hydrophobicN-terminal signal peptide of paraoxonase or derivative thereof,including, but not limited to, human serum paraoxonase/arylesterase(UniProtKB/Swiss-Prot Accession No. P27169.

In some embodiments, a trypanocidal peptide of the present inventionincludes the hydrophobic N-terminal signal peptide of apolipoprotein M(Apo M), including, but not limited to, human apolipoprotein M(UniProtKB/Swiss-ProtO95445), fragments and derivatives thereof.Apolipoprotein M, like haptoglobin-related protein, and paraoxonasedemonstrates an uncleaved signal peptide. For example, trypanocidalpeptides of the present invention include peptides having the N-terminalamino acid residues 1-22, or 2-22 of human apolipoprotein. M(MFHQIWAALLYFYGIILNSIYQ (SEQ ID NO:5) and FHQIWAALLYFYGIILLNSIYQ (SEQ IDNO:6), respectively), fragments, and derivatives thereof. Derivativesinclude, but are not limited to, any of the derivatives describedherein. Derivatives include the exchange of one, two, three, four, ormore hydrophobic amino acid residues for another hydrophobic amino acid.Derivatives include the exchange of one, two, three, four, or morepositively charged amino acid residues for another positively chargedamino acid, including, but not limited to, a substitution at thepositively charged amino acid at position minus 5 from the C terminus.Derivatives may include the deletion of one, two, three, or morehydrophobic amino acids from the known signal sequence. Derivatives mayalso include the deletion of one, two, three, or more very hydrophobicamino acids from the known signal sequence. Derivatives also include anycombination of the above substitutions and/or deletions. Fragments mayinclude, but are no limited to, peptides having at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, or at least 22consecutive amino acids of the hydrophobic N-terminal signal peptide ofapolipoprotein M (Apo M) or derivative thereof, including, but notlimited to, human apolipoprotein M UniProtKB/Swiss-Prot095445.

A trypanocidal peptide of the present invention includes any of thetrypanocidal peptides described herein, including, but not limited to,MSDLGAVISLLLWGRQLFA (SEQ ID NO:1), SDLGAVISLLLWGRQLFA (SEQ ID NO:2),MAKLIATLLGMGLALFRNHQS (SEQ ID NO:3), AKLIATLLGMGLALFRNHQS (SEQ ID NO:4),or SDLGAVISLLWGRQLFA (SEQ ID NO:7), WDLGAVISLLLGGRQLFA (SEQ ID NO:15),SDLGAVIWLLLGGRQLFA (SEQ ID NO:16) or SDLGAVISLLLGGRQLFW (SEQ ID NO:17)and derivatives and fragments thereof. Derivatives include, but are notlimited to, any of the derivatives described herein. Derivativesinclude, but are not limited to, the exchange of one, two, three, four,or more hydrophobic amino acid residues for another hydrophobic aminoacid. Derivatives include, but are not limited to, the exchange of one,two, three, four, or more positively charged amino acid residues foranother positively charged amino acid, including, but not limited to, asubstitution at the positively charged amino acid at position minus 5from the C terminus. Derivatives may also include, but are not limitedto, the deletion of one, two, three, or more very hydrophobic aminoacids from the known signal sequence. Derivatives also include anycombination of the above substitutions and/or deletions. Fragments mayinclude, but are no limited to, peptides having at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, or at least 22consecutive amino acids of SEQ ID NO:1), SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, or SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17.

The present invention provides for the use of trypanocidal peptides as,anti-protozoan agents and provides methods of killing, inhibiting thegrowth, inhibiting the reproduction, and/or rigidifying the plasmamembrane of a protozoan by contacting the protozoan with one or moretrypanocidal peptides. As used herein, the term “inhibit” means prevent,decrease, or reverse. Such contact may be in vitro, ex vivo, and/or invivo. As used herein in vitro is in cell culture, ex vivo is a cell thathas been removed from the body of a subject, and in vivo is within thebody of a subject. As used herein, the term “subject” or “individual”represents an organism, including, for example, a mammal. A mammalincludes, but is not limited to, a human, a non-human primate, livestock(such as, but not limited to, a cow, a horse, a goat, and a pig), arodent, such as, but not limited to, a rat or a mouse, or a domesticpet, such as, but not limited to, a dog or a cat.

The present invention provides methods of killing, inhibiting thegrowth, inhibiting the reproduction, and/or rigidifying the plasmamembrane of a protozoan in a subject by administering to the subject aneffective amount of one or more trypanocidal peptides. A trypanocidalpeptide of the present invention may be administered in an amounteffective to inhibit replication and/or growth of the protozoan. Atrypanocidal peptide of the present invention may be administered in anamount effective to kill a protozoan in an infected individualInhibition of the growth and reproduction of a protozoan and killing ofan a protozoan may be determined by any of various known methods,including, but not limited to, the methods described in the examplesherein.

The present invention provides for the use of trypanocidal peptides asanti-protozoan agents and provides methods of killing, inhibiting thegrowth, inhibiting the reproduction, and/or rigidifying the plasmamembrane of a protozoan by contacting the protozoan with one or moretrypanocidal peptides.

The present invention provides for the use of trypanocidal peptides asplasma membrane rigidifying agents and provides methods for rigidifyingthe plasma membrane of a protozoan by contacting the protozoan with oneor more trypanocidal peptides. In some aspects, the trypanosome is ablood stage form (BSF).

The present invention provides methods of treating or preventing aprotozoan infection in a subject by administering to the subject aneffective amount of one or more trypanocidal peptides. Such atrypanocidal peptide may be identified by the methods described herein.As used herein “treating” or “treatment” may include therapeutic and/orprophylactic treatments. Desirable effects of treatment may includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Atrypanocidal peptide may be administered to a subject to reduce theseverity of the symptoms associated with a protozoan infection. Peptidesof the present invention may be taken as a prophylactic to prevent thedevelopment of a protozoan infection. A peptide of the present inventionmay be administered to a subject to prevent the infection of a subjectwith a protozoan. A peptide of the present invention may be administeredto a subject prior to and/or after exposure to a protozoan.

A trypanocidal peptide may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions andmethods.

By a “therapeutically effective amount” of a trypanocidal peptide ismeant a sufficient amount of the compound to treat the subject at areasonable benefit/risk ratio applicable to obtain a desired therapeuticresponse. It will be understood, however, that the total daily usage ofthe compounds and compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including, for example,the disorder being treated and the severity of the disorder, activity ofthe specific compound employed, the specific composition employed, theage, body weight, general health, sex and diet of the patient, the timeof administration, route of administration, and rate of excretion of thespecific compound employed, the duration of the treatment, drugs used incombination or coincidentally with the specific compound employed, andlike factors well known in the medical arts.

Trypanocidal peptides of the present invention can be administered byany suitable means including, but not limited to, for example,parenteral (involving piercing the skin or mucous membrane), oral(through the digestive tract), transmucosal, rectal, nasal, topical(including, for example, transdermal, aerosol, buccal and sublingual),or vaginal. Parenteral administration may include, for example,subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal,infrasternal, and intraarticular injections as well as various infusiontechniques)

The present invention includes compositions including one or moretrypanocidal peptides as described herein. Also included arecompositions of one or more isolated trypanocidal peptides. As usedherein, the term isolated means a preparation that is either removedfrom its natural environment or synthetically derived, for instance byrecombinant techniques, or chemically or enzymatically synthesized. In apreferred form, an isolated trypanocidal peptides is purified andsubstantially free of other agents.

Compositions may be administered in any of the methods of the presentinvention and may be formulated in a variety of foams adapted to thechosen route of administration. The formulations may be convenientlypresented in unit dosage form and may be prepared by methods well knownin the art of pharmacy. A composition may include a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable,” as usedherein, means that the compositions or components thereof so describedare suitable for use in contact with human skin without undue toxicity,incompatibility, instability, allergic response, and the like. Acomposition may be a pharmaceutical composition.

The preparation of such compositions is well understood in the art. Theformulations of this invention may include one or more accessoryingredients including, but not limited to, diluents, buffers, binders,disintegrants, surface active agents, thickeners, lubricants,preservatives, including, for example, antioxidants, and the like.Pharmaceutically acceptable includes salts, amides and esters that arewell known in the art. Representative acid addition salts include, forexample, hydrochloride, hydrobromide, sulfate, bisulfate, acetate,oxalate, valerate, oleate, palmitate, stearate, laurate, borate,benzoate, lactate, phosphate, toluenesulfonate, methanesulfonate,citrate, maleate, fumarate, succinate, tartrate, ascorbate,glucoheptonate, lactobionate, lauryl sulfate salts, and the like.Representative alkali or alkaline earth metal salts include, forexample, aluminum, calcium, lithium, magnesium, potassium, sodium, orzinc salt, an ammonium salt such as a tertiary amine or quaternaryammonium salt, and an acid salt such as a succinate, tartarate,bitartarate, dihydrochloiide, salicylate, hemisuccinate, citrate,isocitrate, malate, maleate, mesylate, hydrochloride, hydrobromide,phosphate, acetate, carbamate, sulfate, nitrate, formate, lactate,gluconate, glucuronate, pyruvate, oxalacetate, fumarate, propionate,aspartate, glutamate, or benzoate salt, and the like. Pharmaceuticallyacceptable carriers includes, for example, non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Examples of materials that may serveas pharmaceutically acceptable carriers include, but are not limited to,sugars, such as, for example, lactose, glucose and sucrose, starchessuch as, for example, corn starch and potato starch, cellulose and itsderivatives such as, for example, sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate, powdered tragacanth, malt, gelatin,talc, excipients such as, for example, cocoa butter and suppositorywaxes, oils such as, for example, peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil, glycols, such as,for example, propylene glycol, polyols such as, for example, glycerin,sorbitol, mannitol and polyethylene glycol, esters such as, for example,ethyl oleate and ethyl laurate, agar, buffering agents such as, forexample, magnesium hydroxide and aluminum hydroxide, alginic acid,pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcoholand phosphate buffer solutions, as well as other non-toxic compatiblesubstances used in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants such as, for example, sodium lauryl sulfateand magnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants may also be present in the composition,according to the judgment of the formulator.

For parenteral administration in an aqueous solution, the solution maybe suitably buffered if necessary and the liquid diluent first renderedisotonic with sufficient saline or glucose. For enteral administration,the inhibitor may be administered in a tablet or capsule, which may beenteric coated, or in a formulation for controlled or sustained release.Many suitable formulations are known, including polymeric ormicroparticles or nanoparticles encapsulating drug to be released,ointments, gels, or solutions which can be used topically or locally toadminister drug, and even patches, which provide controlled release overa prolonged period of time. These can also take the form of implants.Compositions for nasal administration may be formulated for aerosol orinhalation administration. Such compositions may include solutions insaline which may also contain, for example, benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,and/or other solubilizing or dispersing agents such as those known inthe art. Compositions for rectal administration include, for example,suppositories which may contain a suitable non-irritating excipient,such as cocoa butter, synthetic glyceride esters or polyethyleneglycols, which are solid at ordinary temperatures, but liquify and/ordissolve in the rectal cavity to release the drug.

For human and veterinary administration, compositions of the presentinvention may meet sterility, pyrogenicity, and general safety andpurity standards as required by federal regulatory agencies, such as theFDA. Such compositions are considered suitable for parenteral or enteraladministration to a mammal. Such compositions may be pyrogen-free.

In accordance with the present invention, trypanocidal peptides may beadministered in combination with the administration of one or morepreviously known treatment modalities. As used herein, the term“additional therapeutic agent” represents one or more agents previouslyknown to be effective for the treatment of a protozoan disease or otherconditions. Such an additional therapeutic agent is not a trypanocidalpeptide. The administration of a trypanocidal peptide may take placebefore, during, and/or after the administration of the other mode oftherapy. The present invention includes methods of administering one ormore trypanocidal peptides in combination with the administration of oneor more previously known treatment modalities. The present inventionincludes compositions of one or more trypanocidal peptides and one ormore previously known treatment modalities.

In some embodiments of the present invention, the administration of atrypanocidal peptide in combination with additional therapeutic agentsmay demonstrate therapeutic synergy. Likewise, the administration of twoor more trypanocidal peptides may demonstrate therapeutic synergy. Asused herein, a combination may demonstrate therapeutic synergy if it istherapeutically superior to one or other of the constituents used at itsoptimum dose (Corbett et al., 1982, Cancer Treatment Reports; 66:1187.In some embodiments, a combination demonstrates therapeutic synergy ifthe efficacy of a combination is characterized as more than additiveactions of each constituent.

Liposomes are currently used as drug carriers for a variety of antitumoragents, antiinflammatory agents and the like. The present invention alsoincludes liposomes, lipid carriers, complexes, mixtures, supramolecularstructures, multimolecular aggregates as lipid-based drug deliverysystems including one or more trypanocidal peptides. Such liposomes orliposome-like compositions may be in the form of a monolayer, bilayer,multimolecular aggregate, vesicle, helix, disc, tube, fiber, torus,hexagonal phase, gel phase, liquid-crystalline phase, liquid-crystallinemultimolecular aggregate, micelle, reverse micelle, microemulsion,emulsion, microreservoir, oil globule, fat globule, wax globule and/orcolloidal particle. Such liposomes and liposome-like compositions mayfurther include additional agents, including, for example, additionaltherapeutic agents and/or targeting moieties, including, but not limitedto, RNA apatamers. Such liposomes or liposome-like compositionsincluding one or more trypanocidal peptides may be used in any of themethods described herein.

Compositions of the invention may further include one or more targetingmoieties against parasite target molecules. Compositions including suchtargeting moieties may be used in any of the methods described herein.Targeting moieties include, but are not limited to, high-affinitynucleic acid ligands, also referred to as DNA aptamers and RNA aptamers,that bind with high affinity and high specificity to parasite targetmolecules. For example, a number of trypanosome-specific RNA aptamersthat bind with high affinity and high specificity to the variant surfaceglycoprotein or to an invariant surface domain of live Africantrypanosomes have been identified. See, for example, Goringer et al.,20003, Int J Parasitol; 33(12):1309-17; Lorger et al., 2003, EukaryotCell; 2(1):84-94; Goringer et al., 2006, Handb Exp Pharmacol;(173):375-93; Homann et al., 2006, Comb Chem High Throughput Screen;9(7):491-9; and Adler et al., 2008, Comb Chem High Throughput Screen;11(1):16-23. Any of such RNA aptamers may be used in the compositionsand methods of the present invention.

Many therapeutic agents possess a high degree of hydrophobicity whichcan impede their solubilization in aqueous media and thus hamper theiroral or parenteral administration. Compositions of the present inventionmay include formulations that facilitate the solubilization and/ordelivery of hydrophobic drugs. Such formulations may include any of avariety of such formulations, including, but not limited to, amphiphilicpolymers, lipid-based nanocapsules, nanoformulations, polymericmicelles, magnetic nanocarriers, nano-sized carriers that contain ahydrophobic core, polymeric vectors, lipidic vectors, emulsions, lipidemulsions, and microemulsions. Such formulations may include any of theformulations described herein, such as for example, alcohol and dimethylsulfoxide (DMSO).

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Novel Antimicrobial Peptides Derived from HumanApolipoproteins

This example demonstrates that a small peptide derived from the humanhaptoglobin related protein (Hpr) exhibits potent and specific killingof African trypanosomes. The peptide acts upon the cell membrane of thetrypanosome, inducing a rigidification of the bilayer lipids andsubsequent cell death. The amino acid sequence of the peptide is basedupon the N-terminal signal sequence of Hpr. Hpr is a secreted proteinthat associates with high density lipoproteins (HDL). It is unusual inthat it is secreted without cleavage of its N-terminal signal sequence.Only two other proteins are known that exhibit this phenomenon,paraoxonase 1 and apolipoprotein M, both of which associate with HDL.This small 19 amino acid peptide, based upon the signal sequence of Hprcan be used as a therapeutic agent in the treatment of mammalianpathogens, particularly Trypanosoma brucei, the causative agent ofAfrican sleeping sickness. Currently, there are approximately 500,000infected people in sub-Saharan nations. Treatment of infectedindividuals is limited and there is an alarming increase in the numberof relapses from established drug treatment (Brun et al., 2001, Trop MedInt Health; 6:906-914). Few new drugs are currently under clinicaltrials, and one of the more promising compounds, DB75, has already showna marked tendency to induce drug resistance (Lanteri et al., 2006, MolPharmacol; 70:1585-1592). Based upon the ancient evolutionary origin ofantimicrobial peptides that target cell membranes and their abundance inall branches of life, it is likely that drug resistance will developslowly, if at all, to the peptide of the present invention.

Haptoglobin related protein (Hpr) is a component of the trypanolytichuman high density lipoprotein, termed trypanosome lytic factor (TLF).Unusual for secreted proteins, Hpr retains its N-terminal 19 amino acidsignal peptide (Smith et al. 1995, Science; 268(5208):284-286). Native,delipidated Hpr has been shown to be cytotoxic to Trypanosoma brucei(Shiflett et al., 2005, J Biol Chem; 280(38):32578-32585). This exampleshows that trypanosome killing by Hpr is dependent upon the presence ofthe hydrophobic signal peptide and that a synthetic peptidecorresponding to the signal sequence is sufficient for killing. Indeed,the synthetic Hpr signal peptide (Hpr-SP) specifically killsbloodstream, but not procyclic form T. brucei, is not inhibited by theserum resistance associated protein, which is responsible for evasion ofTLF killing in T. b. rhodesiense (Hager and Hajduk, 1997, Nature;385(6619):823-826), and Hpr-SP appears to act at the surface of thetrypanosome. In vitro studies with model liposomes suggests thatbloodstream form T. brucei are uniquely susceptible to killing by thepeptide due to the acyl chain composition of their cellular membrane.Due to the VSG coat, bloodstream form T. brucei have a high content ofrelatively short myristoyl acyl chains (14 carbon, saturated) (Fergusonet al., 1985, J Biol Chem; 260(27):14547-14555; and Ferguson et al.,1985, J Biol Chem; 260(8):4963-4968), whereas the signal peptideresistant procyclic form T. brucei (the developmental stage found withinthe insect vector) contain a larger content of palmitoyl (16 carbon,saturated) and stearoyl (18 carbon, saturated) chains (Treumann et al.,1997, J Mol Biol; 269(4):529-547), providing for greater van der Waalsinteractions within the bilayer, possibly inhibiting signal peptidepenetration. Data show that the Hpr-SP is not toxic towards mammaliancells, thus it may provide a novel therapeutic for treatment of Africantrypanosomiasis.

Materials and Methods

Peptides. Synthetic peptides corresponding to the 19 amino acidN-terminal signal peptide of Hpr (MSDLGAVISLLLWGRQLFA) (SEQ ID NO:1)were purchased from Bio-Synthesis, Inc. (Lewisville, Tex.).Non-specific, hydrophilic peptides (ERTEESWGRRFWRRGEAC) (SEQ ID NO:10)were predicted from the N-terminus of the alternatively edited protein-1(AEP-1) from mitochondria of T. b. brucei (Ochsenreiter and Hajduk,2006, EMBO; 7(11):1128-1133) and purchased from Alpha Diagnostic (SanAntonio, Tex.).

Lipids. All lipids were purchased from Avanti Polar Lipids (Alabaster,Ala.). These include phosphatidylcholine from egg (#840051) and1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (#850365).

Trypanosome lysis assays. Trypanosomes were cultured in the appropriatemedia and incubated at a cell density of 3×10⁶ cells/ml with theaddition of peptide from a 10 mg/ml stock in DMSO, or control reagentsfor 2 hours (h) at 37° C. Lysis of bloodstream form trypanosomes wasevaluated by phase-contrast microscopy. Procyclic form trypanosomes werestained with 0.1% trypan blue. Trypan blue staining was also used forsome bloodstream form assays to eliminate any possible discrepancybetween the procyclic and bloodstream form assays. No difference wasobserved with the presence of trypan blue in bloodstream form lysisassays.

Mammalian cell viability assays. Human embryonic kidney cells (HEK, ATCC#CRL-1573) were cultured in Eagle's Minimum Essential Mediumsupplemented with 10% fetal bovine serum. Cell viability after treatmentwith Hpr-SP or the relevant controls was determined via trypan blueexclusion. Briefly, cells were plated at approximately 50-60% confluencyinto 96-well poly-lysine coated microtiter plates. Serial dilutions ofHpr-SP from a 10 mg/ml stock in DMSO were added and the cells wereincubated for 2 hours at 37° C. Positive killing controls were serialdilutions of melittin, a membrane permeabilizing peptide from honey beevenom.

Anisotropy assays. Bloodstream form trypanosomes were washed andresuspended at 3×10⁶ cells/ml in phosphate buffered saline. Theanisotropic probe trimethylammonium-1,6-diphenyl-1,3,5-hexatriene(TMA-DPH) was added to a concentration of 0.2 μM and allowed tointercalate into the cell membrane for 15 minutes (min) at ambienttemperature (25° C.). The anisotropic value was acquired with a PerkinElmer Life Sciences fluorescence spectrophotometer LS-55 with excitationat 358 nm and emission read at 430 nm, both with 10 nm slit widths.Values expressed were determined immediately after the addition ofHpr-SP or DMSO only and are the average of three independent assays(except for no addition and 0.167% DMSO). The anisotropy values did notchange over the course of an hour.

Liposome permeabilization assays. Liposomes were constructed byhydration of dried lipid films with 10 mM Hepes and 30 mM calcein to afinal lipid concentration of 10 mg/ml. Resulting multilamellar liposomeswere extruded through a 0.1 polycarbonate filter at temperatures abovethe transition point of the particular lipid mixture. Untapped calceinwas removed by gel filtration (Sephacryl S-300 HR). Permeabilization ofliposomes was monitored fluorimetrically with a Perkin Elmer LS-55luminescent spectrophotometer. Assays were performed by dilutingliposomes 1:1000 into the appropriate buffer and monitoring fluorescencewhen excited by 484 nm light (5 nm slit width) and reading the emissionintensity at 513 nm (10 nm slit widths). The percentage calcein releasewas calculated relative to the 100% fluorescence intensity, achieved bythe addition of 0.01% Triton X-100.

Results

Hpr-SP is sufficient for trypanolysis. Based upon the necessity of theHpr-SP for killing activity by Hpr, the trypanolytic activity of asynthetic peptide corresponding to the 19-amino acids of Hpr-SP wasinvestigated. Addition of Hpr-SP to bloodstream form trypanosomesefficiently killed cells in a dose dependent fashion. The Hpr-SPsolubilizing agent DMSO did not exhibit toxicity at concentrations equalto the highest dosage of Hpr-SP tested. The morphology of Hpr-SP killedtrypanosomes is dramatic and distinct from cells killed by TLF.Excessive fraying of the cellular membrane, and what appears to beleakage of cytoplasmic contents is apparent. These data suggest that themechanism of Hpr-SP trypanosome killing is quite different than thatemployed by TLF. FIG. 1A shows the killing of T. b. brucei by theHpr-SP. Wild type 427 T. b. brucei cells were incubated with increasingconcentrations of Hpr-SP (8-80 μM) added from a stock solution in DMSO.Positive controls were trypanosome lytic factor (TLF). Addition ofequivolume DMSO or equimolar concentrations of a non-specifichydrophilic 19 amino acid peptide (NS 19mer; SEQ ID NO:10) gave notrypanolysis. The inset in FIG. 1A shows cells treated with Hpr-SP. FIG.1B presents a cartoon of Hpr and the derived peptide Hpr-SP (SEQ IDNO:1). Hydrophobic residues are in bold.

Hpr-SP kills SRA-expressing T. brucei and exhibits specificity forbloodstream form trypanosomes. The trypanolytic activity of Hpr-SPagainst procyclic trypanosomes (the insect stage form which dramaticallydown regulates endocytic activity) was assayed. Surprisingly, only veryminor trypanolytic activity was found against these cells (>20% at 80 μMHpr-SP). This discrepancy may be accounted for by the differentcompositions of the bloodstream and procyclic form trypanosomemembranes. Because the Hpr-SP is derived from TLF, we asked whether SRA,the protein responsible for inhibiting TLF killing in the humanpathogenic trypanosome strain T. b. rhodesiense, provided protectionagainst Hpr-SP mediated killing. SRA expressing trypanosomes were killedwith identical activity as non-SRA expressing T. b. brucei. Themorphology of Hpr-SP killed trypanosomes is dramatic and distinct fromcells killed by TLF. Excessive fraying of the cellular membrane, andwhat appears to be leakage of cytoplasmic contents is apparent. Thesedata suggest that the mechanism of Hpr-SP trypanosome killing is quitedifferent than that employed by TLF.

In FIG. 2, lysis assays were conducted with T. b. brucei expressing SRAor procyclic form T. b. brucei. SRA protects human pathogenic T. b.rhodesiense from the lytic activity of TLF. However, SRA offers noprotection against Hpr-SP. Procylic form T. b. brucei are inefficientlykilled by Hpr-Sp. As discussed below, it may be the case that a highercontent of stearoyl acyl chains in the trypanosome membrane protectsagainst the lytic effect of the small highly hydrophobic Hpr-Sp.

Hpr-SP acts at the cytoplasmic membrane of bloodstream formtrypanosomes. A diagnostic feature of TLF killing is the necessity fortrafficking to the lysosome of the target trypanosome, where the acidicenvironment activates a membrane disruptive activity by TLF. Thereforewe asked if compounds that inhibit the acidification of the lysosome(and are known inhibitors of TLF) block the killing activity of Hpr-SP.Thus, lysis assays were performed with Hpr-SP in the presence of 10 mMNH₄Cl or 50 μM chloroquine. No inhibitory effect by these pHneutralizing agents was found, suggesting that either Hpr-Sp isendocytosed but does not require acidic conditions or that the peptideacts at the surface of the target trypanosome. In order to answer thisquestion, lysis assays were performed at 3° C., a temperature thateffectively halts endocytosis. Robust trypanolysis was observed,equivalent to activity observed at 37° C., indicating that the Hpr-SPdoes not require cellular uptake to exert its toxic effects.

In FIG. 3, the site of action was probed by conducting lysis assays inthe presence of chloroquine, NH₄Cl, or at 3° C. Inhibiting theacidification of the lysosomes with either chloroquine NH₄Cl, protectsagainst TLF killing. No inhibition of Hpr-Sp was observed, suggestingthat Hpr-SP does not require targeting tp the intracellular compartment.Endocytosis was inhibited by incubating T. b. brucei at 3° C. The Hpr-SPreadily killed cells at the non-permissive temperature, confirming thecell surface as the site of action of Hpr-Sp.

Hpr-SP exhibits specificity of permeabilizing activity against differentmodel membranes. In order to test the effect of membrane composition onthe specificity displayed by Hpr-SP, a model liposome system wasutilized in which we fluorescently monitored the leakage of dye from theliposome interior. Liposomes composed entirely of egg PC, aheterogeneous mixture with respect to the length and degree ofunsaturations, are readily permeabilized by nanomolar concentrations ofHpr-SP. Permeabilizing activity was robust at pH 6.8, consistent withthe ability of Hpr-SP to target trypanosomal membranes in theextracellular medium. However, when Hpr-SP permeabilizing activityagainst liposomes composed entirely of DSPC, 18-carbon saturated lipids,no permeabilizing activity at neutral or acidic pH was found. Thissuggests that either the Hpr-SP is incapable of intercalating into thehydrophobic regions of the DSPC bilayer or that intercalation of thepeptide is incapable of producing sufficient destabilization, and thuspermeability increase, of the DSCP bilayer.

FIG. 4 shows modeling of T. b. brucei cellular membranes with liposomes.Artificial vesicles were constructed with the phosphatidylcholinescorresponding in acyl chain composition to either the VSG or procyclinlipid anchors, egg phosphatidylcholine (eggPC, transition temperature<0°C.) for VSG and distearoylphosphatidylcholine (DSPS) for procyclin. Theleakage of liposomally entrapped calcein was fluorescently monitored asan indicator of Hpr-SP membrane interaction.

Hpr-SP induces a rigidification of the bilayer lipids in T. brucei. Inorder to understand the effect of Hpr-SP on the cell membrane of targettrypanosomes the lipid bilayer fluidity of trypanosomes treated withHpr-SP was analyzed via anisotropy determinations. The addition of lyticconcentrations of Hpr-SP induced a rigidity increase, i.e. aconstriction of rotational and lateral motion of the lipid components,in the cell membrane. These data are particularly important asbloodstream form trypanosomes rely upon a fluid membrane to recycletheir VSG coat and thus avoid killing mediated by host antibodies.

In FIG. 5, membrane rigidity in live T. b. brucei was monitored with theanisotropic probe TMA-DPH. The addition of Hpr-SP results in an increasein the anisotropy of TMA-DPH labeled cells, indicating membranerigidification. Equivolmes of DMSO, the Hpr-SP solvent, results ineither no change (0.167% DMSO corresponding to 8 μM Hpr-SP) or adecrease (0.4% DMSO corresponding to 20 μM Hpr-SP) in membrane rigidity.Modulation of the membrane fluidity may contribute to the cytotoxity ofHpr-SP.

Hpr-SP is not toxic towards mammalian cells. The potential toxicity ofHpr-SP towards human cells was assayed by determining the effect ofexogenous Hpr-SP on the viability of human embryonic kidney cells. Notoxicity was observed towards these cells by Hpr-SP or equivolumeaddition of DMSO, the solubilizing agent. These data are not unexpectedas Hpr-SP is an endogenously derived molecule that is associated withhepatic cell membranes.

In FIG. 6, cell viability was assayed via trypan blue exclusion. Humanembryonic kidney cells were incubated with increasing concentrations ofHpr-Sp for two hours at 37° C. No cell death was observed withtrypanolytic concentrations of Hpr-SP or equivolume addition of DMSO.Positive killing controls provided by the lytic peptide melittin. Lackof cytotoxicity against mammalian cells suggest that Hpr-SP provide anovel therapeutic strategy for treating African trypanosomiasis.

In conclusion, this example demonstrates that the N-terminal signalsequence of Hpr is sufficient for trypanosome killing; that Hpr-SPexhibits specificity for BSF form T. b. brucei and evades the protectiveeffect of SRA; that Hpr-SP acts at the surface of target trypanosomes,inducing a rigidification of the lipid bilayer; and that the lack oftoxicity towards mammalian cells support Hpr-SP as a therapeutic agent.

Discussion

Previous studies had revealed that human Hpr, a protein encoded by agene that evolved during primate evolution, was toxic to trypanosomesand when assembled in TLF, with another primate specific protein,ApoL-1, had a high specific activity for killing trypanosomes (Shiflettet al., 2005, J Biol Chem; 280(38):32578-32585). Studies to characterizethe mechanism of Hpr killing revealed that Hpr was unusual in that itcontained an unprocessed N-terminal signal peptide (Hpr-SP). Deletion ofthe Hpr-SP inactivated recombinant Hpr (Vanhollebeke et al., 2007, PNAS;104(10):4118-4123). The 19 amino acid Hpr-SP was synthesized and testedfor trypanosome killing activity. Hpr-SP damage to the trypanosomes isstriking. Cells rapidly fragment and the rate of killing is independentof temperature. On the other hand, TLF killing of trypanosomes requiresendocytosis and lysosomal trafficking, has a characteristic lag phase of˜20 min and killing activity is completely blocked at temperatures below4° C. When human embryonic kidney cells were incubated at the sameconcentration of Hpr-SP no toxicity was observed, indicating the Hpr-SPselectively interacts with trypanosomes.

These results show that Hpr-SP is highly toxic to trypanosomes, at lowμM concentrations, and acts rapidly at the cell surface. In addition,mammalian cells are resistant to Hpr-SP. The rigidification increaseseen upon addition of Hpr-SP to bloodstream form trypanosomes may bedirectly involved in the cytotoxicity of the peptide. T. brucei rapidlyturn over a dense protein surface coat through endocytosis at a specificsite at the posterior of the cell (Engstler et al., 2007, Cell;131(3):505-515). This turnover facilitates evasion from the host immunesystem. The fluidity of the cell membrane has been directly implicatedin this process as the proteins are swept backwards towards theendocytic site by outside physical forces, i.e. the flow of theextracellular environment. In this regard it may even be the case thatthe peptide will facilitate immune destruction of trypanosomes byblocking the turnover of the cellular protein coat.

Example 2 Plasma Membrane of Bloodstream Form African TrypanosomesConfers Susceptibility and Specificity to Killing by HydrophobicPeptides

The developmental stage of T. brucei found within the mammalian host,the bloodstream form (BSF), is a highly motile cell (Rodriguez et al.,2009, PNAS; 106(46):19322-19327; and Oberholzer et al., 2010, PLoSPathog; 6(1):e1000739), exhibits rapid rates of endocytosis (Engstler etal., 2004, J Cell Sci; 117(Pt 7):1105-1115) and free diffusion ofdensely packed GPI-anchored proteins (Bulow et al., 1988, Biochem;27(7):2384-2388). Endocytosis is restricted to the flagellar pocket, asmall, specialized membrane structure at the posterior of the cell(Field and Carrington, 2009, Nature Rev; 7(11):775-786). This morphologyrequires that surface associated cargo destined for endocytosis belaterally sorted in the plane of the membrane to the flagellar pocket.Perhaps the most striking example of this activity is the sorting ofimmunoglobulin bound variant surface glycoproteins (VSG) to theflagellar pocket by physical forces generated from the flow ofextracellular medium over the surface of the trypanosome (Engstler etal. Al., 2007, Cell; 131(3):505-515). The nature of the trypanosomeplasma membrane that facilitates rapid lateral sorting is not known. Inthe case of VSG, surface flow may benefit from the use of myristate, arelatively short acyl chain, as the membrane-anchoring moiety (Fergusonet al., 1985, J Biol Chem; 260(27):14547-14555; and Ferguson et al.,(1985), J Biol Chem, 260(8):4963-4968). The procyclic form (PCF) T.brucei, found within the insect midgut, does not exhibit rapid rates ofendocytosis (Langreth and Balber, 1975, J Protozool; 22(1):40-53; andMorgan et al., 2001, J Cell Sci; 114(Pt 14):2605-2615) and theGPI-anchored procyclin surface proteins are anchored with longerpalmitoyl and stearoyl acyl chains (Field et al., 1991, EMBO;10(10):2731-2739).

The structure of the plasma membrane is also important for avoidinglysis by host defense factors such as complement or antimicrobialpeptides. The terminal components of the complement membrane attackcomplex are sterically hindered from assembling by the dense surfacecoat of VSG. Human defensins, antimicrobial peptides with a distincttertiary structure, are relatively inefficient at killing BSF T. brucei(McGwire et al., 2003, J Infect Dis; 188(1):146-152) and it may be thecase that the VSG coat inhibits interaction with the cell membrane.Interestingly BSF and PCF T. brucei exhibit differential susceptibilityto a number of antimicrobial peptides (McGwire et al., 2003, J InfectDis; 188(1):146-152). This distinction may be attributed to thedifferent surface density of GPI-anchored proteins between the twoforms, VSG being present at roughly an order of magnitude greater thanprocylin, thereby providing greater steric hinderance. Alternatively, orin addition to the difference in surface protein density, thedifferences in phospholipid and sterol composition between the twodevelopmental forms may play a role.

The present example describes the trypanocidal activity of a smallhydrophobic peptide (SHP-1), derived from the signal sequence of a humanapolipoprotein, haptoglobin related protein (Hpr), which circumventssteric hinderance from the VSG coat and interacts with the plasmamembrane of BSF T. brucei. This example shows that this non-cationicpeptide specifically targets fluid membranes. The SHP-1 rapidly bindsBSF cells rather than PCF or mammalian cells. Consistently, BSF T.brucei are readily killed by SHP-1 and no lysis or killing is observedwith PCF T. brucei, mammalian cells or human erythrocytes.

Methods

Peptides. Synthetic peptides corresponding to the N-terminal signalpeptide of human apolipoproteins haptoglobin related protein (“SHP-1,”SDLGAVISLLLWGRQLFA (SEQ ID NO:2)), paraoxonase-1 (“SHP-2,”(AKLIATLLGMGLALFRNHQS (SEQ ID NO:4)) (both without the N-terminalmethionine residue) and all derivatives (“SHP-1-ΔL,” SDLGAVISLLWGRQLFA(SEQ ID NO:7); “SHP-1-ΔLLL,” SDLGAVISWGRQLFA (SEQ ID NO:8); and“SBP-1-ΔLGA,” SDVISLLLWGRQLFA (SEQ ID NO:9) were purchased fromBio-Synthesis, Inc. (Lewisville, Tex.). A non-specific, hydrophilicpeptide (ERTEESWGRRFWRRGEAC (SEQ ID NO:10)) predicted from theN-terminus of the alternatively edited protein-1 from mitochondria of T.b. brucei, was purchased from Alpha Diagnostic (San Antonio, Tex.)(Ochsenreiter and Hajduk, 2006, EMBO; 7(11):1128-1133).

Lipids. All lipids were purchased from Avanti Polar Lipids (Alabaster,Ala.). These include phosphatidylcholine from egg (egg PC, #840051),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (#850365),1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (#850360),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (#850355),1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (#850350),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (#850345),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (#850457) and(#850467).

Trypanosome killing assays. Bloodstream form T. b. brucei Lister 427(MiTat 1.2), T. b. gambiense type 1 (Eliane strain) and T. b.rhodesiense KETR12482 were used in these studies. Trypanosome killingassays were performed as described previously (Hajduk et al., 1989, JBiol Chem; 264(9):5210-5217; Shiflett et al., 2005, J Biol Chem;280(38):32578-32585; and Widener et al., 2007, PLoS Pathog;3(9):1250-1261). Cultured trypanosomes (except for T. b. gambiense whichwere incubated at 1×10⁶ cells/ml) were incubated in HMI-9 media (BSF) orSM media (PCF) at a density of 1×10⁷ cells/ml containing either 10% heatinactivated fetal bovine serum or bovine serum albumin and peptide orcontrol reagents for two hours at 37° C. Killing of BSF trypanosomes wasevaluated by phase-contrast microscopy. Procyclic form trypanosomes werestained with 0.1% trypan blue. Trypan blue staining was also used forsome BSF assays to eliminate any possible discrepancy between the PCFand BSF assays. Trypan blue was added at the endpoint of each assay toavoid any toxic effects. No difference was observed with the presence oftrypan blue in BSF killing assays.

Mammalian cell viability and hemolysis assays. Human embryonic kidneycells (HEK) (ATCC CRL-1573) and LNCaP prostate cancer cells (ATCCCRL-1740) were utilized for cell viability assays. Human embryonickidney cells were cultured in Dulbecco's Modified Eagles Medium, highglucose (Thermo Scientific, cat #SH30243.01) with 10% fetal bovine serumat 37° C. and 5% CO₂. Prostate cancer cells were cultured in RPMI-1640medium (Invitrogen, cat #A10491-01) with 10% fetal bovine serum at 37°C. and 5% CO₂. In both cases, assays were performed by aliquoting cellsinto 96 well plates at approximately 60% confluency and allowing thecells to adhere for two hours. Cells were then incubated with serialdilutions of SHP-1, or the relevant control, in the corresponding mediafor two hours at 37° C. Cell viability was determined by the ability oflive cells to exclude trypan blue. The cells were incubated with 0.1%trypan blue for 10 minutes and examined microscopically for cytoplasmicstaining. The potential for SHP-1 to induce hemolysis was assayed bymonitoring hemoglobin (Hb) release from freshly collected humanerythrocytes that had been washed and incubated with SHP-1 in PBS. TheHb concentration of the supernatant was determined by the absorbance at412 nm and compared to 100% hemolysis acquired by hypotonic lysis. Themembrane permeabilizing peptide, melittin, was used a positive controlfor killing or hemolysis.

Microscopy. All images were acquired with an Axio Observer Z1 equippedwith an Axiocam MRm controlled by the Axiovision 4.6 software or a ZeissImager A1. Fluorescent microscopy with Texas Red-labelled SHP-1 wasperformed by incubating 3×10⁶ cells/ml with 8 μM TR-Hpr-SP in media for10 min before fixing with 1% paraformaldehyde for 1 min, air drying onglass slides and covering with DAPI containing antifade reagentProlongGold (Molecular Probes). Videos were acquired with live cells ata density of 3×10⁶ cells/ml incubated with the indicated concentrationof SHP-1 at 37° C. Movies were recorded at 63× magnification at 50 msecacquisition times for normal and constricted motion trypanosomes and 25msec acquisition times for hyperactivated trypanosomes. Visualizedtrypanosomes presented in supplementary movies were centered in thevideos by digital tracking with Final Cut software (Apple) and videoswere looped to facilitate comparisons.

Flow cytometry. Peptide binding to cells was monitored by flowcytometry. Binding assays were performed with 3×10⁶ cells/ml in PBS at25° C. Texas red-labelled SHP-1 was added to a final concentration of0.8 μM and 50,000 cells were immediately counted with the CyAn ADP flowcytometer (Dako, available on the worldwide web at dako.com). Data wereanalyzed with FlowJo software (Treestar, available on the worldwide webat treestar.com).

Calcein release assays. Permeabilization of unilamellar liposomes wasassayed as described previously (Harrington, et al., 2009, J Biol Chem;284(20):13505-12). Briefly, liposomes were diluted 1:1000 into 50 mMTris, pH 7.0 and calcein fluorescence was monitored at 513 nm whenexcited at 484 nm. The percent calcein release was calculated relativeto the 100% fluorescence intensity, achieved by the addition of 0.01%Triton X-100.

Anisotropy assays. The membrane fluidity of live T. b. brucei was probedby measuring the fluorescence depolarization of1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatrienep-toluenesulfonate (TMA-DPH) (Invitrogen, cat. #T204). Cells were washedtwice with, and resuspended in PBS, or Voorheis's modified PBS(supplemented with 10 mM glucose, 79 mM sucrose) in the case ofprocyclic cells, at a density of 3×10⁶ cells/ml. The anisotropic probe,TMA-DPH, was added to a final concentration of 0.5 μM and allowed tointercalate into the cell membrane for 15 min. Anisotropic values wereacquired via the software function of the Perkin Elmer Life SciencesLS55. Samples were excited at 358 nm and emission was read at 430 nm,both with 10 nm slit widths. Data was corrected for light scatteringwith an unlabeled sample of cells and anisotropy was calculatedaccording to the equation, r=(I_(VV)−GI_(VH))/(I_(VV)+2GI_(VH)), where ris the anisotropy value, I_(VV) is the emission intensity acquired withthe excitation and emission polarizing filters set vertically, G is theinstrument correction factor and I_(VH) is the emission intensityacquired with the excitation polarizing filter set vertically and theemission polarizing filter set horizontally. All assays were conductedat ambient temperature, ˜25° C.

Results

Small hydrophobic peptides specifically kill BSF African trypanosomes.IT has been previously reported that delipidated native human Hpr,purified from HDL, kills BSF T. b. brucei (Shiflett et al., 2005, J BiolChem; 280(38):32578-32585; and Smith et al. 1995, Science;268(5208):284-286). The lack of killing activity from a recombinant formof Hpr that lacks the N-terminal signal peptide (Vanhollebeke et al.,2007, PNAS; 104(10):4118-4123) led us to investigate the potentialtrypanocidal activity of a synthetic peptide corresponding to the signalsequence. Addition of SHP-1 to BSF T. b. brucei killed cells in a dosedependent fashion (FIG. 7A). Equivolume amounts of DMSO did not exhibittoxicity. The human pathogenic subspecies T. b. rhodesiense and T. b.gambiense are also sensitive to killing by SHP-1 (FIG. 7A). Cellstreated with lower concentrations of SHP-1, ie. 4 μM, retain theirelongated and twisted shape, and cell membranes appear completelyintact, however cells are motionless and unable to exclude trypan blue(FIG. 7B). At higher concentrations of SHP-1, 40-80 μM, BSF trypanosomesretain their overall shape, however cellular membranes appear to havebeen stripped off or collapsed into the cytoplasm of the cells (FIG.7B). Membrane degradation occurs subsequent to cell death, as revealedby video DIC microscopy.

SHP-1 exhibits specificity for BSF African trypanosomes. The specificityof SHP was initially examined by conducting killing assays with PCF T.b. brucei. No killing of PCF cells was observed (FIG. 7A). Flowcytometry was utilized to determine if the lack of PCF killing was dueto a failure of SHP-1 to bind the PCF trypanosome (FIG. 7C). Texas-redSHP-1 rapidly binds BSF T. brucei as indicated by the labeling of theentire population of cells immediately after the addition of peptide. Nolabeling of PCF cells was observed immediately after addition of peptideindicating a dramatic difference in the affinity of SUP-1 for BSF andPCF trypanosomes.

The analysis of the spectrum of SHP-1 toxicity was broadened by testingthe peptide against two human cell lines. Incubating human embryonickidney cells (HEK) or prostate cancer cells (LNCaP) with relatively highconcentrations of SHP-1, 80-160 μM, for 2 h at 37° C. did not result incell death as evaluated by changes in morphology or the ability toexclude trypan blue (FIGS. 8A and 8B). Consistent with a lack oftoxicity, flow cytometry indicated that these cells are not bound bySHP-1 (FIG. 8C). Additionally no hemolysis of freshly collected humanerythrocytes incubated with 240 μM SHP-1, the highest concentrationtested, was detected (FIG. 8D).

SHP-1 killing of BSF trypanosomes requires a hydrophobic stretch ofamino acids. The sequence requirements for trypanocidal activity bySUP-1 (SEQ ID NO:2) were analyzed by conducting killing assays withsynthetic peptides that mimicked, lessened or ablated the corehydrophobic region of SHP-1 (FIG. 9A). A peptide exhibiting similarhydrophobicity and length was derived from the signal sequence of thehuman apolipoprotein paraoxonase-1 (SHP-2) (SEQ ID NO:4) (Sorenson,1999, Arterioscler Thromb Vasc Biol; 19(9):2214-2225). Trypanocidalactivity was observed against BSF T. brucei equivalent to SHP-1 (FIG.9B). Conversely, a non-specific hydrophilic peptide of equal length toSHP-1 exhibited no toxicity (FIG. 9B). The size of the hydrophobic coreof SHP-1 was decreased by a single leucine deletion from the C-terminalleucine triplicate (SEQ ID NO:7). Decreasing the length andhydrophobicity by a single amino acid resulted in a five-fold decreasein trypanocidal activity (FIG. 9B). Further deletions of the C-terminalleucine triplicate (SEQ ID NO:8) or the N-terminalleucine-glycine-alanine (SEQ ID NO:9) resulted in a complete lose oftrypanosome killing activity (FIG. 9B).

The effect of altering peptide hydrophobicity on trypanosome killingsuggested that membrane interaction might play a mechanistic role inSHP-1's trypanotoxicity. In an initial attempt to address thispossibility, a model liposome system was utilized in which the releaseof internally trapped fluorophore, calcein, was monitored as anindicator of membrane interaction. The SHP-1 elicits calcein releasefrom unilamellar liposomes composed of phosphatidylcholine (PC) from eggat nanomolar concentrations (FIG. 9C). Variant SHP peptides performed inthe calcein release assay in a manner consistent with their ability tokill BSF trypanosomes, with a lack of liposome permeabilization by thedeletion variants. The SHP-2 elicited calcein release, albeit reducedfrom SHP-1, while deleting a single leucine, the leucine truplicate orthe leucine-glycine-alanine stretch resulted in a loss of permeabilizingactivity at concentrations of 500 nM, the highest concentration tested(FIG. 9C).

These data indicate that, the amino acid sequence of SHP-1 is notstrictly required but the necessary characteristic for trypanosomekilling activity is a significantly high degree of hydrophobicity, andthe ability to interact with lipid bilayers is consistent with theability to kill BSF trypanosomes.

SHP acts at the surface of BSF trypanosomes. Multiple trypanocidalmolecules have been identified which exert their toxic effect afterlocalization within an intracellular vesicle rather than at the cellsurface (Hager et al., 1994, J Cell Biol; 126(1):155-167; and Delgado etal., 2009, Cell Death Differ; 16(3):406-416). Therefore, whether SHP-1requires internalization by the target trypanosome was addressed.Killing assays were performed with SHP-1 at 3° C., a temperature thathalts endocytosis (Hager et al., 1994, J Cell Biol; 126(1):155-167).Robust trypanocidal activity (FIG. 10A) was observed, equivalent tokilling assays performed at 37° C., suggesting that SHP-1 does notrequire cellular uptake to exert its toxic effects. Consistent withthese data, Texas Red-labeled SHP-1 uniformly binds the surface of BSFtrypanosomes at 37° C. rather than accumulating in intracellularvesicles (FIG. 10B).

SHP-1-membrane interaction is dependent on lipid bilayer fluidity. Basedupon the physiological necessity of membrane surface flow in BSF T.brucei, the role lipid bilayer fluidity plays in dictating sensitivityto SHP-1 was addressed. Liposomes composed of highly fluid compositionssuch as egg phosphatidylcholine (PC) (Tm<0° C.) (Koynova and Caffrey,1998, Biochim Biophys Acta; 1376(1):91-145), are readily permeabilizedby SHP-1 (FIG. 11A). However, homogenous liposomes composed entirely ofsymmetric PC with saturated chains of 15 to 18 carbon atoms (Tm 34° C.and 55° C. respectively) (Koynova and Caffrey, 1998, Biochim BiophysActa; 1376(1):91-145) are not permeabilized by SHP-1 (FIG. 11A).Fluidizing refractory lipid compositions by incorporating increasingmole percent DMPC (symmetric 14:0, Tm=23.6° C.) led to aDMPC-concentration-dependent increase in susceptibility to SHP-1 (FIG.11B).

To determine whether SBP-1 has a specific affinity for membranemyristate or susceptibility is due to a general physical, propertyimparted by the short acyl chain, liposomes composed of PC withdifferent acyl chain moieties and transition temperatures (Tm) wereassayed. The presence of an unsaturation in 16- or 18-carbon acyl chainsresults in a large decrease in bilayer transition temperature relativeto their unsaturated counterparts. Liposomes composed of POPC, Tm=−2° C.(25), or SOPC, 18:0-18:1 PC, Tm=6° C. (25), are susceptible topermeabilization by SHP-1 indicating that myristate is not a requirementfor membrane interaction (FIG. 11C). The role of membrane fluidity inmediating membrane sensitivity to SHP-1 was further investigated byconducting permeabilization assays with the refractory lipidcompositions at temperatures above their Tm. Fluidizing lipid bilayerscomposed of 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (symmetric15:0, Tm=34° C.) or DPPC (symmetric 16:0, Tm=41.3° C.) (Koynova andCaffrey, 1998, Biochim Biophys Acta; 1376(1):91-145) by bringingliposomal suspensions above their respective Tm resulted in sensitivityto permeabilization by SHP-1 (FIG. 11D). These data indicate that it isnot affinity for a specific bilayer moiety but the general physicalproperty of membrane fluidity that confers susceptibility to SHP-1.

SHP-1 induces rigidification of BSF trypanosome cell membranes. Basedupon the role of lipid bilayer fluidity in SHP-1 membrane interactionand the restriction of liposomal acyl chain motion by E. coli LamBsignal sequence peptides (Jones and Gierasch, (1994) Biophys J;67(4):1534-1545), the effect SHP-1 has upon the fluidity of BSF T.brucei membranes was assayed. The surface membrane probe TMA-DPH, acationic lipophilic molecule that rapidly partitions into the outerleaflet of the cellular lipid bilayer, was utilized. Addition of SHP-1increases the rigidity of BSF cell membranes as indicated by an increasein the anisotropy of the surface membrane probe TMA-DPH (FIG. 12A). Thedifference in membrane composition of PCF trypanosomes is apparent inthe higher anisotropic values, i.e. greater rigidity, acquired for PCFcells. Consistent with the lack of SHP-1 binding to PCF cells and datafrom model liposomes indicating that a high degree of fluidity isrequired for SHP-1 intercalation, the anisotropy of PCF trypanosomes isunchanged by the addition of peptide. In contrast to BSF cells, PCFtrypanosomes are significantly less motile and do not exhibit highturnover rates of surface proteins or endocytic activity.

SHP-1 induces dramatic changes in cell motility. During the course ofour investigations on BSF T. brucei killing, it readily became apparentthat treatment of cells with SHP-1 resulted in motility changes. Inorder to assess the effect of SHP-1 on the motility of trypanosomes BSFcells were analyzed by DIC video microscopy. Addition of 8 M SHP-1resulted in a progression of motility changes over 30 min (FIG. 12B).Untreated BSF trypanosomes exhibit a characteristic corkscrew motion(FIG. 12C). Within a minute of SHP-1 addition, a significant fraction ofcells exhibit hyperactive motility (FIG. 12C). Hyperactivated cells arestill present at 10-20 minutes, however, a portion of cells displayhighly constricted motion (FIG. 12C). The constricted motility phenotypeis increasingly apparent at 20 minutes, with approximately 50% of thecells displaying constriction, the other 50% remaining hyperactivated intheir motion. During the time course dead cells are increasinglyapparent. This phenomenon is more pronounced at 80 μM SHP-1 with all ofthe cells exhibiting hyperactivity until slowing down and dying within10 min.

Discussion

This example demonstrates that a small peptide that rapidly intercalatesinto the plasma membrane of BSF T. brucei and induces cell death. Thepeptide is highly specific for the developmental form found within amammalian host. Specificity is mediated at the level of binding, or morelikely intercalation into the acyl chain region of the target membrane.Indeed, studies with model membrane systems indicate that the peptide issensitive to the acyl chain composition of lipid bilayers. In particularthe membrane fluidity that is imparted by a given acyl chain compositiondictates the ability of this peptide to interact with membranes.

Killing of BSF T. brucei has been demonstrated for a variety of bonafide antimicrobial peptides (McGwire et al., 2003, J Infect Dis;188(1):146-152; and Haines et al., 2009, PLoS Negl Trop Dis; 3(2):e373),such as the cathelicidins and their derivatives novispirin andovispirin, as well as unusual candidates such as neuropeptides (Delgadoet al., 2009, Cell Death Differ; 16(3):406-416). In the case ofcathelicidins it has been shown that these peptides permeabilize theplasma membrane and cells assume a rounded and crumpled morphology upondeath (McGwire et al., 2003, J Infect Dis; 188(1):146-152). Atrelatively high concentrations of SHP-1, massive disruption of thecellular membrane is observed, however, as revealed by video microscopy,this occurs subsequent to cell death. Treatment with lowerconcentrations of SHP-1, i.e. 4 μM, results in dead T. brucei thatexhibit a normal morphology and DIC microscopy indicates that thecellular membrane remains intact. The lack of osmotic swelling suggeststhat permeabilization of the plasma membrane is not the mechanism ofkilling. Furthermore, the observed changes in BSF T. brucei cellmembrane rigidity by SBP-1 suggest a novel mechanism of toxicity.

Bloodstream form African trypanosomes are highly dynamic cells withrespect to motility (Rodriguez et al., 2009, PNAS; 106(46):19322-19327;and Oberholzer et al., 2010, PLoS Pathog; 6(1):e1000739), endocytosisand vesicle trafficking (Engstler et al., 2004, J Cell Sci; 117(Pt7):1105-1115) and lateral flow of surface molecules (Bulow et al., 1988,Biochem; 27(7):2384-2388; and Engstler et al., 2007, Cell;131(3):505-515). All of these activities require movement of membranecomponents such as phospholipids, and in the case of endocytosis andvesicle trafficking, remodeling of the lipid bilayer. Decreasing thephysical property of fluidity in the bulk membrane such that theseactivities are burdened may contribute to a general poisoning of thecell by SBP-1. Alternatively, or in synergy with membranerigidification, SBP-1 may act through non-specific alterations ofintegral membrane protein stability/activity (Lee, 2004, Biochim BiophysActa; 1666(1-2):62-87). For example, distortion of the lateral pressureprofile, a mechanism that has been attributed to general anesthetics(Cantor, 1998, Toxicol Lett; 100-101:451-458), may lead to increasedlateral pressure which in turn may physically inhibit transmembranechannels (Kamaraju and Sukharev, 2008, Biochem; 47(40):10540-10550).Additionally, peptides corresponding to the E. coli LamB signal peptidehave been shown to effect the oligomerization of integral transmembraneproteins (Benach et al., 2003, J Biol Chem; 278(6):3628-3638). Suchsurface activity is consistent with the SHIP-1 site of action being theplasma membrane as suggested by killing assays performed at 3° C. (FIG.10A) and labeling of the surface of BSF T. brucei by Texas-red-SHP-1(FIG. 10B). In either case, the fluidity of the BSF trypanosome cellmembrane appears to not only be modified by the SHP-1, but alsospecifically targeted.

The studies of this example, with model liposomes, indicate that thefluidity of the target lipid bilayer determines the ability of SHIP-1 tobind and permeabilize membranes. These data are consistent with previousstudies showing that modulation of the surface pressure of lipidmonolayers dictates the ability of signal peptides to intercalate intothe acyl chain region (Briggs et al., 1986, Science; 233(4760):206-208).Membranes exhibiting gel phase lipid order are refractory topermeabilization by SHP-1. When these refractory liposomes are broughtabove their transition temperatures, thus exhibiting liquid crystallineorder, they are rendered susceptible to permeabilization by SHP-1. Thesedata indicate that the lateral van der Wants forces dictate the abilityof SHP-1 to intercalate into target lipid environments. The immediatelabeling of the entire population of BSF T. brucei by Texas-red labeledSHP indicates a very rapid rate of bilayer intercalation (FIG. 7C).

The consequences of SHP-1 intercalation into BSF trypanosome membranesare immediately apparent on the cellular level. Cells exhibithypermotility immediately after introduction of SHP-1 subsequentlybecoming constricted in their motion before dying. The cause for thesechanges in motility can only be speculated upon. Spermatozoa areexamples of flagellated cells that undergo changes in membrane fluidityconcurrent with the onset of hypermotility (Visconti et al., 2002, JReprod Immunol; 53(1-2):133-150). Hyperactivation is associated withefflux of cholesterol and an increase in membrane fluidity. In thisregard Tyler et al. have recently demonstrated that T. brucei flagellarmembranes are enriched in cholesterol and exhibit a higher degree oforder than the pellicle of the cell (Tyler et al., 2009, J Cell Sci;122(Pt 6):859-866). It is intriguing to imagine that SHP-1 induces aredistribution of cholesterol from the flagellum to the pellicularfraction of the cell membrane. This would result in an overall increasein cell membrane rigidity coupled with a localized decrease of theflagellar rigidity, and thus alleviation of physical strain againstflagellar motors and acceleration of flagellar beating. The observedconstricted motility at later time points following treatment with SHP-1may be the result of metabolic depletion.

Small hydrophobic peptides target a fundamental physiologicalcharacteristic of T. brucei, namely the fluid physical property of thecell membrane. It is not likely that BSF trypanosomes will quicklydevelop a strategy to circumvent the cytotoxicity of molecules thatalter the mechanoelastic properties of the cell membrane. This exampledemonstrates that the BSF trypanosome cell membrane is an attractivetarget for the development of novel therapeutics. See also, Harringtonet al., 2010, J Biol Chem, 285(37):28659-66 (Epub 2010 Jul. 8).

Example 3 Killing T. congolense, T. vivax, and the Metacyclic Forms ofT. brucei

African trypanosomes are eukaryotic parasites that cause sleepingsickness in humans and a wasting disease known as Nagana in cattle. Asshown in the previous examples, a small trypanocidal peptide has beenderived from the human trypanosome lytic factor (TLF), a subset of highdensity lipoproteins that confers immunity to veterinary pathogenictrypanosomes. As described in Examples 1 and 2, the peptide kills boththe veterinary pathogen Trypanosoma brucei brucei and the humanpathogenic subspecies responsible for African sleeping sickness, T. b.rhodesiense and T. b. gambiense. Screening of the peptide against anumber of eukaryotic pathogens, human cell lines and the procyclic formtrypanosome, the developmental stage within the insect vector midgut,indicates that the peptide uniquely targets the developmental stage oftrypanosome found within the mammalian host. The lack of toxicityagainst mammalian cell lines suggests a therapeutic potential fortreatment of human and animal trypanosomiasis. This example will testthe peptides against the major cattle pathogens T. congolense and T.vivax. Additionally, the activity of the peptide will be tested againstmetacyclic trypanosomes, the developmental form within the tsetse flysalivary gland and thus the first developmental stage encountered by apotential human or animal host.

Trypanosome lytic factor uniquely contains two trypanolytic proteins,haptoglobin related protein (Hpr) and apolipoprotein L-1 (Shiflett etal., 2005, J Biol Chem; 280(38):32578-32585). Haptoglobin relatedprotein is unusual in that it is secreted from hepatocytes withoutcleavage of its hydrophobic N-terminal signal peptide (Hpr-SP) (Smith etal., 1995, Science; 268(5208): 284-286). Previous work established thathuman Hpr, when purified in a delipidated form, kills bloodstream formT. b. brucei (Shiflett et al., 2005, J Biol Chem; 280(38): 32578-32585).The lack of trypanolytic activity from recombinant Hpr (Vanhollebeke etal. 2006, PNAS; 104(10), 4118-4123) led us to investigate the potentialactivity of a synthetic 19-amino acid peptide corresponding in sequenceto the Hpr-SP. The previous examples have established that Hpr-SP istoxic to all bloodstream form T. brucei and that the specificity ofHpr-SP is mediated by the high presence of myristate, a short 14 carbon,saturated acyl chain, in the cell membrane. The unusually high contentof myristate is due to the glycerophosphoinositol-anchoring of thetrypanosome's variable surface glycoproteins. The high content ofmyristate lends the cell membrane a high degree of fluidity thatfacilitates both the rapid uptake of bound host antibodies and cellsurface bound nutrients. The Hpr-SP induces a rigidification of the cellmembrane that results in dramatic motility changes and rapid onset ofcell death (as shown, for example, in Examples 1 and 2).

In the case on of the T. brucei subspecies, the membrane composition isparticularly well characterized; The VSG proteins in bloodstream formtrypanosomes are membrane anchored via myristate (Ferguson et al., 1985,J Biol Chem; 260(27):14547-14555), whereas the procyclic forms utilizelonger saturated chains, stearate and palmitate (Field et al., 1991,EMBO; 10(10): 2731-2739) which provide increased lateral van der Waalsforces and presumably protect the membrane from intercalation of Hpr-SP.Example 2 shows that cells refractory to Hpr-SP killing are not bound bythe peptide.

With this example, the cattle pathogens T. congolense and T. vivax willbe tested for susceptibility to binding and killing by hydrophobicsignal sequence peptides, including, but not limited to, SEQ ID NO:1-20.There are some indications that these species possess different membranecompositions than the T. brucei subspecies. For instance probing thesurface with antibodies against surface lectins suggests a less denseVSG coat, and therefore less myristate within the cell membrane. Invitro killing assays will be carried out against T. congolense and T.vivax. Assays will be performed by incubating trypanosomes with variousconcentrations of peptide in HMI-9 media for two hours at 37° C.Purified human TLF will serve as a positive killing control.Additionally, the binding, or lack thereof, of hydrophobic signalsequence peptides to these cell lines will be determined utilizingTexas-red labeled Hpr-SP and analysis by flow cytometry or fluorescencemicroscopy.

The metacyclic form of T. brucei resides within the salivary glands ofthe tstese fly vector. This developmental stage is primed forencountering a potential mammalian host through the bite of the fly.Consistently, the metacyclic form expresses GPI-anchored VSG rather thanthe procyclic acid repeats characteristic of the developmental stageswithin the mid- and hind gut of the tsetse fly. Thus, the metacyclicforms will likely be susceptible to killing by Hpr-SP. Metacyclic T.brucei will be collected from tsetse fly salivary glands, washed intoPBS supplemented with 30% media and subject to the previously describedassays. These assays will indicate the possibility of utilizinghydrophobic signal sequence peptides, such as Hpr-SP, as a moleculecapable of preventing initial infection of potential animal hosts frominteraction with the trypanosome containing insect vector. The studiesof this example will greatly inform our knowledge of the therapeuticpotential of Hpr-SP and the membrane physiology of different Trypanosomasp. and developmental forms.

Example 4 Novel African Trypanocidal Agents Membrane RigidifyingPeptides

The bloodstream developmental forms of pathogenic African trypanosomesare uniquely susceptible to killing by small hydrophobic peptides.Trypanocidal activity is conferred by peptide hydrophobicity and chargedistribution and results from increased rigidity of the plasma membrane.This mechanism reveals a necessary phenotype, high membrane fluidity,unique to these pathogens, and indicates that the plasma membrane andits biosynthetic components are novel targets for the development ofpharmaceutical agents.

As shown in the previous examples, both veterinary and human pathogenicbloodstream form (BSF) T. brucei are uniquely susceptible to killing bytwo small hydrophobic peptides, SHP-1 and SHP-2. This exampledemonstrates that the specificity of SHP is mediated by a high degree offluidity in the plasma membrane of BSF cells. These peptides do notbind, and thus do not kill, procyclic (PC) form T. brucei, which has amore rigid plasma membrane, or mammalian cells, nor are they hemolyticat concentrations orders of magnitude higher than necessary to kill BSFT. brucei. See also, Harrington et al., 2010, J Biol Chem;285(37):28659-66.

Materials and Methods

Peptides and Lipids. All peptides were purchased from Bio-Synthesis,Inc. (Lewisville, Tex.). All lipids were purchased from Avanti PolarLipids (Alabaster, Ala.). These include phosphatidylcholine from egg(8450051) and1-palmitoyl-2-(6,7-dibromo)stearoyl-sn-glycero-3-phosphocholine (850480)and 1-palmitoyl-2-(9,10-dibromo)stearoyl-sn-glycero-3-phosphocholine(850481).

Trypanosome Killing Assays. Light microscopy based trypanosome killingassays were performed as previously described in detail (Harrington etal., 2010, J Biol Chem; 285(37):28659-66; Widener et al., 2007, PLoSPathog; 3(9):1250-61; Shiflett et al., 2007, J Eukaryot Microbiol;54(1):18-21; and Hajduk et al., 1989, J Biol Chem; 264(9):5210-7).Metacyclic T. b. brucei TREU 667 were obtained from dissection of thesalivary glands of infected tsetse flies. Newly hatched Glossinamorsitans morsitans (24-48 hours post eclosion) were fed defibrinatedhorse blood containing 2×10⁶ ml⁻¹ trypanosomes and maintained at 25° C.in 75% relative humidity and fed maintenance blood meals three times perweek. Following day 20-30, flies were harvested and salivary glands weredissected out and washed in HMI 9 media containing 10% fetal bovineserum. Metacyclic cells were collected via centrifugation and maintainedin HMI 9 media with 10% fetal bovine serum at 37° C. in 5% CO₂ untiluse. Killing assays were conducted with 1×10⁴ cells/ml in HMI 9 mediacontaining 10% fetal bovine serum. Trypanosoma vivax and T. congolensewere grown from stabilites in donor ICR mouse (Harlan, United Kingdom).Parasites were harvested from mice by terminal exsanguination andpassage of infected blood through a small anion exchange column. Cellswere maintained in HMI 9 with 20% goat serum at 37° C. in 5% CO₂ untiluse. Killing assays were performed with 1×10⁷ cells/ml in HMI 9 mediawith either 20% goat serum, in the case of T. vivax and T. congolense,and 10% fetal bovine serum, for T. b. brucei. Cells were incubated at37° C. for 2 hours and dead parasites were scored visually. All wereconducted in at least duplicate, and data points are the averages withstandard deviations.

Anisotropy Assays. The plasma membrane rigidity of live T. b. brucei wasdetermined by measuring the fluorescence depolarization ofdiphenyl-1,3,5-hexatriene p-toluenesulfonate (DPH) or1-(4-trimethylammoniumphenyl)-6-diphenyl-1,3,5-hexatrienep-toluenesulfonate (TMA-DPH; Invitrogen T204). Cells were washed 3 timeswith and resuspended in PBS at a density of 3×10⁶ cells/ml. Theanisotropic probes were added to a final concentration of 0.5 M andallowed to intercalate into the cell membrane for one hour in the dark.Anisotropic values were acquired via the software function of aPerkinElmer Life Sciences LS55 spectrofluorometer. Samples were excitedat 358 nm, and emission was read at 430 nm, both with 10-nm slit widths.Temperature was maintained at 37° C. by means of the PerkinElmer LS55Biokinetics accessory. Data were corrected for light scattering with anunlabeled sample of cells, and anisotropy was calculated according tothe equation r=(I_(VV)−GI_(VH))/(I_(VV)+2GI_(VH)), where r is theanisotropy value, I_(VV) is the emission intensity acquired with theexcitation- and emission-polarizing filters set vertically, G is theinstrument correction factor, and I_(VH) is the emission intensityacquired with the excitation-polarizing filter set vertically and theemission-polarizing filter set horizontally. All assays were conductedat 37° C. Data points shown are the average of triplicate measurementswith standard deviations.

Trypanosome motility. All images and videos were acquired with an AxioObserver Z1 equipped with an AxioCam MRm controlled by AxioVision 4.6software. Videos were acquired with live cells at a density of 1×10⁷cells/ml in HMI 9 media with 10% fetal bovine serum, incubated with 40 MSHP-1 at 37° C. Videos were recorded at magnification 63× with 50-msacquisition times. The motility of BSF trypanosomes was scored visuallyfrom video playback of trypanosomes scanned throughout 10 μl aliquots.Data is shown as the average of triplicate trials with standarddeviations.

Parallax Analysis. The hydrocarbon penetration depth of tryptophansspaced throughout synthetic peptides corresponding to SHP-1 or SHP-3(Table 1) was determined by parallax analysis with brominatedphosphatidylcholine liposomes. Large unilamellar liposomes composed ofegg phosphatidylcholine and 10 mol %1-palmitoyl-2-(6,7-dibromo)stearoyl-sn-glycero-3-phosphocholine (shallowquencher) or1-palmitoyl-2-(9,10-dibromo)stearoyl-sn-glycero-3-phosphocholine (deepquencher) were constructed by hydration of a thin dry lipid film withphosphate buffered saline. Resulting multilamellar liposomes were madeunilameller via extrusion through polycarbonate filters with 0.1 μmpores. Peptides (500 nM) were incubated with 200 g/ml liposomes inphosphate buffered saline at 37° C. for 22 hours (h). Tryptophanfluorescence at 357 nm was measured from at least triplicate trials inthe PerkinElmer Life Sciences LS55 spectrofluorometer and an excitationwavelength of 280 nm for SHP-1 and 290 nm for SHP-2, 10 nm excitationand 9 nm emission slit widths. The distance of tryptophans from thebilayer center (ZCF) is calculated from the equation:Z_(CF)=L_(C1)+[−ln(F₁/F₂)/πC−L₂₁ ^(2]/)2L₂₁ (Chattopadhyay and London,1987, Biochemistry; 26(1):39-45); where L_(C1) is the distance from thecenter of the bilayer to the shallow quencher, in this case 10.8 Å for6,7-dibromo-PC (McIntosh and Holloway, 1987, Biochemistry;26(6):1783-8), F₁ is the intensity of tryptophan in the presence of theshallow quencher and F₂ is the tryptophan intensity in the presence ofthe deep quencher, C is the mole fraction of quencher divided by thearea of individual phospholipid (70 Å2), and L₂₁ is the difference inthe depth of the two quenchers (2.7 Å) (McIntosh and Holloway, 1987,Biochemistry; 26(6):1783-8). The hydrocarbon insertion depth oftryptophans is then given by one half the bilayer thickness, 29 Å(McIntosh and Holloway, 1987, Biochemistry; 26(6):1783-8), minus Z_(CF).

Flow Cytometry. Peptide binding to BSF T. b. brucei was monitored byflow cytometry. Binding assays were performed with 3×10⁶ cells/ml in HMI9 plus 10% fetal bovine serum at 25° C. FITC-labeled SHP-1 or SHP-3 wasadded to a final concentration of 8 μM and 50,000 cells were immediatelycounted on a CyAn ADP flow cytometer (Dako). Data were analyzed withFlowJo software (TreeStar Inc.).

Calcein Release Assays. Membrane permeabilization assays were conductedas described in detail previously (Harrington et al., 2010, J Biol Chem;285(37):28659-66; and Harrington et al., 2009, J Biol Chem;284(20):13505-12). Unilamellar liposomes were constructed as describedabove but with 30 mM calcein in 10 mM Hepes as the hydration buffer.Untrapped dye was removed by gel (Sephacryl S-300 HR, GE Healthcare).Liposomes were diluted 1:1000 into phosphate buffered saline, andcalcein fluorescence was monitored at 513 nm when excited at 484 nm. Thepercent calcein release was calculated relative to the 100% fluorescenceintensity, achieved by the addition of 0.01% Triton X-100.

Discussion

An immediately apparent difference between the plasma membranes of BSFand PC African trypanosomes is the lack of a dense coat of VSG in theinsect stage cells. Metacyclic stage cells, which do express a VSG coat,were tested for susceptibility to SHP-1 and found no killing activity(FIG. 13A). Next this example determined whether other Africantrypanosomes are sensitive to SHP. Bloodstream developmental forms ofboth T. vivax and T. congolense are susceptible to killing by SHP-1 atconcentrations similar to BSF T. brucei (FIG. 13A), indicating thatmembrane fluidity is a characteristic of both human and veterinarypathogenic African trypanosomes.

As shown in the previous examples, trypanocidal SHP are derived fromapolipoproteins and exhibit the characteristics of secretory signalpeptides, i.e. size (18-22 amino acids), a central hydrophobic regionand a C-terminal putative signal peptidase cleavage site defined byspecific amino acid patterns. See also Harrington et al., 2010, J BiolChem; 285(37):28659-66. Although these peptides share physical features,their primary structures are entirely different (FIG. 13B). A third,distinct SHP (SHP-3) was tested for trypanocidal activity, also derivedfrom an apolipoprotein (Axler et al., 2008, FEBS Lett; 582(5):826-8) andpossessing similar features as SHP-1 and SHP-2 (FIG. 13C). Despitepossessing the same general physical characteristics and binding to BSFT. brucei (FIG. 16), no killing was seen against BSF T. brucei (FIG.13C). Comparison of the three sequences revealed that an arginine atposition −5 relative to the C-terminus is common to trypanocidal SHP-1and SHP-2, but is absent in SHP-3 (FIG. 13B). Substitution of anarginine for the leucine in this position of SHP-3 (SHP-3ΔR, FIG. 13B)confers trypanocidal activity (FIG. 13C). Replacement of the leucinewith glutamate in SHP-3 (SHP-3ΔE, FIG. 13B) does not (FIG. 13C).Trypanolytic SHP-2 has a positive charge at both the N- and C-terminus,SHP-1 has only a single positive charge at the C-terminus; thus whethercharge location is important was tested by swapping the C-terminalasparagine with the N-terminal aspartate of SHP-1 (SHP-1-swap, FIG.13B). Rearranging the residues resulted in a loss of trypanocidalactivity (FIG. 13C).

As shown in the previous examples, trypanocidal SHP act at the plasmamembrane but do not induce osmotic swelling or bursting (see also,Harrington et al., 2010, J Biol Chem; 285(37):28659-66), suggesting thatany effect upon the BSF trypanosome must not result in a loss of plasmamembrane integrity. This example investigated the rigidity of BSF T.brucei membranes, a property that can change without loss of membraneintegrity, utilizing two anisotropic probes, diphenylhexatriene (DPH)that reports on the interior of the acyl chain region, andtrimethylammonium-diphenylhexatriene (TMA-DPH) that is anchored at themembrane interface. Addition of either trypanocidal or non-trypanocidalSHP to BSF T. brucei results in increased rigidity of the interiorregion of the plasma membrane (FIG. 14A). However only the trypanocidalSHP-1, SHP-3 and SHP-3ΔR, increased the rigidity of the interfacialregion of the plasma membrane (FIG. 14B). These data indicate thatrigidification of the interfacial region is necessary for SHP killing ofBSF T. brucei. The requirement for a C-terminal charge may indicate thation pairing with the phospholipid headgroups contributes to an increasein interfacial rigidity and thus cell death.

Treatment of BSF African trypanosomes with SHP results in multiplephysiological alterations. Rigidification of the plasma membrane bySHP-1 has a direct effect, decreasing the fraction of VSG exhibitinglateral mobility (FIG. 14C). Another physiological consequence, that mayor may not be related to membrane rigidification, is SHP-induced changesin cell motility. As shown in Example 2, SHP-1 causes an initialhyperactivation followed by constricted motility and cell death (seealso Harrington et al., 2010, J Biol Chem; 285(37):28659-66).Non-trypanocidal SHP-3 does cause initial hyperactivation of BSF T. b.brucei, however subsequent constriction of motility does not occur (FIG.14D). Trypanocidal SHP including the SHP-3 variant, SHP-3ΔR, induce bothinitial hyperactivation and subsequent constriction (FIG. 14D).Constricted motility may result in reduced hydrodynamic forces actingupon surface proteins. Therefore trypanocidal SHP may not only directlykill BSF African trypanosomes, but may also render them more susceptibleto immune killing by delaying the clearance of surface bound hostdefense molecules (Engstler et al., 2007, Cell; 131(3):505-15).

In order to understand why trypanocidal and non-trypanocidal SHP havedifferential effects on the BSF plasma membrane, this example determinedthe orientation of SHP-1 and SHP-3 in lipid bilayers by parallaxanalysis (Chattopadhyay and London, 1987, Biochemistry; 26(1):39-45).Tryptophans were substituted at positions 1, 8 and 18 (N- to C-terminus,native tryptophan located at position 12) (Table 1) in SHP-1 andpositions 1, 13 and 20 in SHP-3 (N- to C-terminus, native tryptophanlocated at position 5) (Table 1). These placements were chosen, andnative tryptophan residues were replaced with glycine, in order toretain the hydrophobic profile of the original peptides.

TABLE 1 Sequences and Quenching Data of SHP Tryptophan Variants. PeptideSequence (N- to C- terminus)¹ SEQ ID No: F₁/F₂ ± S.D.² SHP-1ΔW1WDLGAVISLLLGGRQLFA 15 0.8897 ± 0.1112 SHP-1ΔW8 SDLGAVIWLLLGGRQLFA 160.9767 ± 0.0717 SHP-1 SDLGAVISLLLWGRQLFA  2 0.9676 ± 0.0560 SHP-1ΔW18SDLGAVISLLLGGRQLFW 17 0.9285 ± 0.1557 SHP-3ΔW1 WHQIGAALLYFYGIILNSIY 18N.A. SHP-3 FHQIWAALLYFYGIILNSIY 11 0.9202 ± 0.1260 SHP-3ΔW13FHQIGAALLYFYWIILNSIY 19 0.9912 ± 0.0787 SHP-3ΔW20 FHQIGAALLYFYGIILNSIW20 1.1620 ± 0.3194 ¹Peptide sequences illustrate the different positionsof tryptophan substitutions (bold). ²Quenching data is presented as theratio of tryptophan fluorescence intensity in the presence of theshallow, F₁, and deep, F₂, quencher and the standard deviations (S.D.).

All of the substituted SHP-1 peptides show equivalent killing activityas well as membrane interaction (FIGS. 17A and 17B). The insertion depthof SHP tryptophans was determined by measuring the quenching efficiencyof egg phosphatidylcholine liposomes containing 10 mol % brominatedphospholipid. Ratiometric analysis of the quenching efficiency ofbromines located at the 6,7 position and 9,10 positions of the acylchains indicates that SHP-1 does not penetrate deeply into thehydrocarbon region and adopts a shallow U-shaped conformation (FIG. 15,Table 1). The two terminal tryptophans, positions 1 and 18, are locatedapproximately 1.1 and 2.0 Å from the membrane interface respectively.The internal tryptophans at positions 8 and 12 are located approximately7.8 and 5.1 Å from the interface respectively. Therefore, rather thanaligning with the phospholipid acyl chains, SHP-1 inserts into theexterior leaflet parallel to the plane of the bilayer and proximal tothe interface with the aqueous environment. These data are consistentwith what has been observed for other signal peptides (Jones andGierasch, 1994, Biophys J; 1994. 67(4):1534-45; and Voglino et al.,1999, Biochemistry; 38(23):7509-16). Non-trypanocidal SHP-3 and thetryptophan variants also exhibit membrane interaction with modelliposomes (FIG. 17C). Parallax analysis of SHP-3 indicates deeperpenetration into the hydrocarbon chains and a tilted orientation (FIG.15). The N-terminal tryptophan was inefficiently quenched, suggestingthat it does not intercalate into the hydrocarbon region. The nativetryptophan, position 5, inserts to approximately 1.6 Å below theinterface, while the tryptophan at position 13 is located approximately4.7 Å deep and the C-terminal tryptophan penetrates most deeply, toapproximately 11.2 Å. This orientation precludes interaction of theC-terminus with the lipid headgroups. These data again suggest a rolefor the positively charged C-terminus in mediating the interfacialrigidity increase of the outer leaflet of the plasma membrane.

African trypanosomes present an attractive target for membranerigidifying peptides. The BSF cells exhibit extremely high rates ofendocytosis and recently it has been reported that nanobodies againstVSG that block endocytosis are highly efficient trypanolytic agents(Stijlemans et al., 2011, PLoS Pathog; 7(6):e1002072). Surfaceassociated cargo, such as Ig-bound VSG are laterally sorted in themembrane, potentially over the entire length of the cell (Engstler etal., 2007, Cell; 131(3):505-15). Membrane rigidification may contributeto cell death by hindering these activities. A fluid membrane may alsofacilitate the diffusion of small molecule nutrients into the BSF cell.Finally, it has been shown that increasing the rigidity of trypanosomemembranes results in a redistribution of proteins normally localized tothe flagellar membrane (Tyler et al., 2009, J Cell Sci; 122(Pt6):859-66). Therefore trypanolytic SHP may have pleiotropic effects,interfering with any number of these physiological processes. Thespecificity of SHP for BSF African trypanosomes reveals a phenotype thatmay be taken advantage of for the development of pharmaceutical agents.Drugs that target the fluidity of the plasma membrane may offer a meansof circumventing the rapid onset of resistance exhibited by thesepathogens. Compensating for a phenotype that is the result of a systemof gene products would require multiple viable mutations rather than asingle mutation within a targeted enzyme or transporter. Additionallythe peptides described herein represent a tool to investigate themolecular basis of membrane fluidity.

Example 5 Efficacy of Small Hydrophobic Peptides (SHP) Against AfricanTrypanosome Infection in Mice

Peptides of the present invention may be tested in animal models,including, but not limited to, mouse model systems, foranti-trypanosomal activity.

Injection of parasites into animals. Mice will be injected with adefined number of Trypanosoma brucei brucei maintained as frozen stocksin liquid nitrogen (0.1-0.2 mL of infected animal blood with DMSO (finalconcentration 7.5%)). Stocks will be thawed at 37° C., and diluted withan equal volume of sterile saline containing 1% glucose. Mice will beinfected by peritoneal injection of parasites. Animals will be swabbedwith 70% ethanol on the abdomen prior to injection. Small gauge needles(for example, 26 g) will be used. Animals will be securely held and thehypodermic needle slowly inserted approximately 0.5 cm through the skininto the abdomen at a 60° angle. Parasites will be injected slowly intothe abdomen and the needle slowly withdrawn. Following injection,animals will be released into a clean cage and monitored for abnormalactivity. The site of injection will be examined five minutes postinjection for swelling, bleeding, or other abnormal signs.

Administration of SHP. SHP will be administered by intraperitonealinjection as described above. Peptide will be diluted from a 20 mM stockin DMSO into sterile phosphate buffered saline. Several dosagesencompassing a range of 15-60 mg/kg will be administered daily.Alternatively peptide will be administered via oral corn oil gavage.

Monitoring of parasite numbers in infected animals. Numbers of T. b.brucei will be monitored daily by phase contrast microscopy of bloodsamples. This procedure causes only slight and momentary pain to theanimal. Tails will be first treated with 2% lidocaine as an analgesic. Adrop of blood will be obtained by clipping the extreme end of the mousetail. 10-20 μl of blood will be milked from the tail vein directly ontoa microscope slide for examination. Snipped tails will be treated withliquid bandage to seal the wound and animals will be observed for woundclosure. Infections will be monitored for 2-7 days. Parasitemias will bemonitored daily and animals will be observed for appearance andbehavior. Animals exhibiting signs of stress, ruffled coat, slow orerratic movement will be euthanized immediately. Under no circumstanceswill animals be allowed to suffer from parasite infection.

Pre-toxicity screens in non infected mice using up to 100 mg/kg may alsobe undertaken.

Further, guidelines of the World Health Organization (WHO) parasite drugdiscovery initiative may be followed for the identification anddevelopment of new drug candidates (reviewed, for example, by Nwaka andHudson, 2006, Nat Rev Drug Discov; 5:941-95, Nwaka and Ridley, 2003, NatRev Drug Discov; 2:919-928, and Nwaka et al., 2009, PLoS Negl Trop Dis;3(8):e440).

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Sequence Listing Free Text

-   SEQ ID NO:1 N-terminal amino acid residues 1-19 of the human    haptoglobulin-related protein-   SEQ ID NO:2 N-terminal amino acid residues 2-19 of the human    haptoglobulin-related protein (SHP-1)-   SEQ ID NO:3 N-terminal amino acid residues 1-22 of the human    paraoxonase-1 protein-   SEQ ID NO:4 N-terminal al amino acid residues 2-22 of the human    paraoxonase-1 protein (SHP-2)-   SEQ ID NO:5 N-terminal amino acid residues 1-22 of the human    apolipoprotein M (Apo M)-   SEQ ID NO:6 N-terminal amino acid residues 2-22 of the human    apolipoprotein M (Apo M)-   SEQ ID NO:7 Derivative of SHP-1 having a single leucine deletion-   SEQ ID NO:8 Derivative of SHP-1 with deletion of C-terminal leucine    triplicate-   SEQ ID NO:9 Derivative of SHP-1 with a deletion of the N-terminal    leucine-glycine-alanine residues-   SEQ ID NO:10 Non-specific, hydrophilic peptide predicted from the    N-terminus of the alternatively edited protein-1 from mitochondria    of T. b. brucei-   SEQ ID NO:11 Amino acid sequence of small hyrophobic peptide 3    (SHP-3)-   SEQ ID NO:12 Derivative of SHP-3 having a leucine to arginine    substitution at position −5-   SEQ ID NO:13 Derivative of SHP-3 having a leucine to glutamic acid    substitution at position −5-   SEQ ID NO:14 Derivative of SHP-1 having an aspartic acid to arginine    substitution at position 2 and an arginine to aspartic acid    substitution at position −5-   SEQ ID NO:15 Derivative of SHP-1 having a tryptophan to glycine    substitution at position 1-   SEQ ID NO:16 Derivative of SHP-1 having a tryptophan to glycine    substitution at position 8-   SEQ ID NO:17 Derivative of SHP-1 having a tryptophan to glycine    substitution at position 18-   SEQ ID NO:18 Derivative of SHP-3 having a tryptophan to glycine    substitution at position 1-   SEQ ID NO:19 Derivative of SHP-3 having a tryptophan to glycine    substitution at position 13-   SEQ ID NO:20 Derivative of SHP-3 having a tryptophan to glycine    substitution at position 20

What is claimed is:
 1. A method of inducing rigidification of the plasmamembrane of a bloodstream form of a kinetoplastid protozoan of the genusTrypanosoma, the method comprising contacting the protozoan with anisolated hydrophobic signal sequence peptide, wherein the isolatedhydrophobic signal sequence peptide consists of 17 to 25 amino acidresidues of an uncleaved, hydrophobic N-terminal signal sequence,wherein the isolated hydrophobic signal sequence peptide contains apositively charged amino acid at position minus five relative to theC-terminus of the hydrophobic signal sequence peptide, and wherein thehydrophobic signal sequence peptide comprises at least nine consecutiveamino acid residues of MSDLGAVISLLLWGRQLFA, SEQ ID NO:1 or a derivativeof SEQ ID NO:1, wherein the derivative of SEQ ID NO:1 has up to fourhydrophobic amino acid residues of SEQ ID NO:1 exchanged for anotherhydrophobic amino acid, and/or one positively charged amino acidresidues of SEQ ID NO:1 exchanged for another positively charged aminoacid.
 2. The method of claim 1, wherein the hydrophobic signal sequencepeptide is soluble in ethanol or dimethyl sulfoxide (DMSO).
 3. A methodof inducing rigidification of the plasma membrane of a bloodstream formof a kinetoplastid protozoan of the genus Trypanosoma, the methodcomprising contacting the protozoan with an isolated hydrophobic signalsequence peptide, wherein the isolated hydrophobic signal sequencepeptide consists of 17 to 25 amino acid residues of an uncleaved,hydrophobic N-terminal signal sequence, wherein the isolated hydrophobicsignal sequence peptide contains a positively charged amino acid atposition minus five relative to the C-terminus of the hydrophobic signalsequence peptide, and wherein the hydrophobic signal sequence peptidecomprises at least nine consecutive amino acid residues of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17. 4.The method of claim 1, wherein the trypanosome is selected from thegroup consisting of Trypanosoma brucei brucei, T. b. gambiense, and T.b. rhodesiense, T. congolense, and T. vivax.
 5. The method of claim 1,wherein the hydrophobic signal sequence peptide is provided as acomposition further comprising liposome, emulsion, or micelle.
 6. Themethod of claim 1, wherein the hydrophobic signal sequence peptide isprovided as a composition further comprising an RNA aptamer that bindsto a structurally conserved region of a trypanosome variant surfaceglycoprotein (VSG).
 7. The method of claim 1, wherein the hydrophobicsignal sequence peptide is provided as a pyrogen-free composition. 8.The method of claim 1, wherein the plasma membrane rigidificationresults in killing, inhibition of growth, inhibition of reproduction,plasma membrane degradation, and/or constricted cell motility of thebloodstream form of a kinetoplastid protozoan of the genus Trypanosoma.9. The method of claim 1 wherein contacting the protozoan with ahydrophobic signal sequence peptide comprises in vitro, ex vivo, or invivo contact.
 10. The method of claim 9 comprising in vivo contactwithin the body of a mammalian subject.
 11. A method of inducingrigidification of the plasma membrane of a bloodstream form of akinetoplastid protozoan of the genus Trypanosoma, the method comprisingcontacting the protozoan with an isolated hydrophobic signal sequencepeptide, wherein the isolated hydrophobic signal sequence peptide isselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:7, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.
 12. A method ofinducing rigidification of the plasma membrane of a bloodstream form ofa kinetoplastid protozoan of the genus Trypanosoma, the methodcomprising contacting the protozoan with an isolated trypanocidalpeptide of 12 to 25 amino acid residues in length, wherein the isolatedtrypanocidal peptide comprises at least nine consecutive amino acidresidues of MSDLGAVISLLLWGRQLFA, SEQ ID NO:1 or a derivative of SEQ IDNO:1, wherein the derivative of SEQ ID NO:1 has up to four hydrophobicamino acid residues of SEQ ID NO:1 exchanged for another hydrophobicamino acid, and/or one positively charged amino acid residues of SEQ IDNO:1 exchanged for another positively charged amino acid.
 13. The methodof claim 3, wherein the plasma membrane rigidification results inkilling, inhibition of growth, inhibition of reproduction, plasmamembrane degradation, and/or constricted cell motility of thebloodstream form of a kinetoplastid protozoan of the genus Trypanosoma.14. The method of claim 3, wherein the trypanosome is selected from thegroup consisting of Trypanosoma brucei brucei, T. b. gambiense, and T.b. rhodesiense, T. congolense, and T. vivax.
 15. The method of claim 3,wherein the hydrophobic signal sequence peptide is provided as acomposition further comprising liposome, emulsion, or micelle.
 16. Themethod of claim 3, wherein the hydrophobic signal sequence peptide isprovided as a composition further comprising an RNA aptamer that bindsto a structurally conserved region of a trypanosome variant surfaceglycoprotein (VSG).
 17. The method of claim 3, wherein the hydrophobicsignal sequence peptide is soluble in ethanol or dimethyl sulfoxide(DMSO).
 18. The method of claim 3, wherein the hydrophobic signalsequence peptide is provided as a pyrogen-free composition.
 19. Themethod of claim 3, wherein contacting the protozoan with a hydrophobicsignal sequence peptide comprises in vitro, ex vivo, or in vivo contact.20. The method of claim 19 comprising in vivo contact within the body ofa mammalian subject.
 21. The method of claim 11, wherein the plasmamembrane rigidification results in killing, inhibition of growth,inhibition of reproduction, plasma membrane degradation, and/orconstricted cell motility of the bloodstream form of a kinetoplastidprotozoan of the genus Trypanosoma.
 22. The method of claim 11, whereinthe trypanosome is selected from the group consisting of Trypanosomabrucei brucei, T. b. gambiense, and T. b. rhodesiense, T. congolense,and T. vivax.
 23. The method of claim 11, wherein the hydrophobic signalsequence peptide is provided as a composition further comprisingliposome, emulsion, or micelle.
 24. The method of claim 11, wherein thehydrophobic signal sequence peptide is provided as a composition furthercomprising an RNA aptamer that binds to a structurally conserved regionof a trypanosome variant surface glycoprotein (VSG).
 25. The method ofclaim 11, wherein the hydrophobic signal sequence peptide is soluble inethanol or dimethyl sulfoxide (DMSO).
 26. The method of claim 11,wherein the hydrophobic signal sequence peptide is provided as apyrogen-free composition.
 27. The method of claim 11, wherein contactingthe protozoan with a hydrophobic signal sequence peptide comprises invitro, ex vivo, or in vivo contact.
 28. The method of claim 27comprising in vivo contact within the body of a mammalian subject. 29.The method of claim 12, wherein the plasma membrane rigidificationresults in killing, inhibition of growth, inhibition of reproduction,plasma membrane degradation, and/or constricted cell motility of thebloodstream form of a kinetoplastid protozoan of the genus Trypanosoma.30. The method of claim 12, wherein the trypanosome is selected from thegroup consisting of Trypanosoma brucei brucei, T. b. gambiense, and T.b. rhodesiense, T. congolense, and T. vivax.
 31. The method of claim 12,wherein the hydrophobic signal sequence peptide is provided as acomposition further comprising liposome, emulsion, or micelle.
 32. Themethod of claim 12, wherein the hydrophobic signal sequence peptide isprovided as a composition further comprising an RNA aptamer that bindsto a structurally conserved region of a trypanosome variant surfaceglycoprotein (VSG).
 33. The method of claim 12, wherein the hydrophobicsignal sequence peptide is soluble in ethanol or dimethyl sulfoxide(DMSO).
 34. The method of claim 12, wherein the hydrophobic signalsequence peptide is provided as a pyrogen-free composition.
 35. Themethod of claim 12, wherein contacting the protozoan with a hydrophobicsignal sequence peptide comprises in vitro, ex vivo, or in vivo contact.36. The method of claim 35 comprising in vivo contact within the body ofa mammalian subject.